Oligonucleotide lipid nanoparticle compositions, methods of making and methods of using the same

ABSTRACT

Methods for inhibiting oligonucleotide activity in vitro or in vivo to a cell that are formulated with at least one oligonucleotide encapsulated in a lipid nanoparticle are disclosed.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 14/678,589filed Apr. 3, 2015, which claims priority to U.S. ProvisionalApplication 61/975,366, filed Apr. 4, 2014, the disclosure of which ishereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was not made with government support and the governmenthas no rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Apr. 3, 2015, is named604_56088_SEQ_LIST_OSIF-2014-218.txt, and is 19,311 bytes in size.

TECHNICAL FIELD

The present disclosure pertains to lipid nanoparticles (LNPs) usable forthe delivery of therapeutic compositions, including, but not limited tonucleic acids (NAs), and to the methods of making the same. In aparticular aspect, described herein are quaternary-tertiary lipoaminesystems useful for the delivery of anti-miRNA oligonucleotides.

BACKGROUND OF THE INVENTION

Nucleic acid (NA)-based therapies using miRNAs (miRs) and/or anti-miRNAare being developed to promote or inhibit gene expression. miRNAs arenon-coding RNAs that can regulate expression of networks of genes by themechanism of RNA interference (RNAi). This occurs by incorporation ofmiRNA into an RNA-induced silencing complex (RISC) that mediatetranslational repression and degradation of target mRNAs. An antimiR isan oligonucleotide that is complementary to a miRNA that inhibits itsfunction thereby de-represses the target genes of the miRNA. Asmutations in genes and changes in miRNA profile are believed to be theunderlying cause of cancer and other diseases, NA-based agents candirectly act upon the underlying etiology, maximizing therapeuticpotential. Non-limiting examples of NA-based therapies include: plasmidDNA (pDNA), small interfering RNA (siRNA), small hairpin RNA (shRNA),miR mimic (mimetic), anti-miR/antagomiR/miR inhibitor, and antisenseoligonucleotide (ASO). Until the development of the nanoparticlecompositions described herein, the clinical translation of NA-basedtherapies faced several obstacles in their implementation sincetransporting NAs to their intracellular target was particularlychallenging and since NAs are relatively unstable and subject todegradation by serum and cellular nucleases. Further, the high negativecharges of NAs made it impossible for diffusion across the cellmembrane, further limiting utility.

A liposome is a vesicle composed of one or more lipid bilayers, capableof carrying hydrophilic molecules within an aqueous core or hydrophobicmolecules within its lipid bilayer(s). As used herein, “lipidnanoparticles” is a general term to described lipid-based particles inthe submicron range. Lipid nanoparticles can have structuralcharacteristics of liposomes and/or have alternative non-bilayer typesof structures. Drug delivery by lipid nanoparticles via systemic routerequires overcoming several physiological barriers. Thereticuloendothelial system (RES) is responsible for clearance of lipidnanoparticles from the circulation. Once escaping the vasculature andreaching the target cell, lipid nanoparticles are typically taken up byendocytosis and must release the drug into the cytoplasm prior todegradation within acidic endosomes and lysosomes.

In particular, the delivery of such nucleic acids (NAs), including siRNAand other therapeutic oligonucleotides is a major technical challengethat has limited their potential for clinical translation.

The development of efficient delivery vehicles is a key to clinicaltranslation of oligonucleotide (ON) therapeutics. It is desired that alipid nanoparticle formulation should be able to (1) protect the drugfrom enzymatic degradation; (2) transverse the capillary endothelium;(3) specifically reach the target cell type without causing excessiveimmunoactivation or off-target cytotoxicity; (4) promote endocytosis andendosomal release; and (5) form a stable formulation with colloidalstability and long shelf-life.

SUMMARY OF THE INVENTION

Provided herein are lipid nanoparticles that can encapsulate therapeuticoligonucleotides with high efficiency and fulfill physical andbiological criteria for efficacious delivery.

In one broad aspect, there is provided herein a composition comprisingat least one formulated modified anti-miR-21 oligonucleotide comprisinga nucleic acid sequence complementary to the sequence of miR-21, whereinthe nucleic acid sequence may include chemical modifications such asphosphorothioate linkages and 2′-O-methoxyethyl modifications.

In another broad aspect, there is provided herein a method for loweringthe biological activity of miR-21 in vitro comprising: i) contacting abiological sample expressing miR-21 therein, with a formulated modifiedanti-miR-21 oligonucleotide, wherein the anti-miR-21 oligonucleotidecomprises a nucleic acid sequence complementary to the sequence ofmiR-21; and, ii) lowering the biological activity of miR-21 in vitro.

In another broad aspect, there is provided herein a method of loweringthe biological activity of miR-21 in vivo in a subject comprising: i)administering to the subject a formulated anti-miR-21oligonucleotide,wherein the anti-miR-21 oligonucleotide comprises a nucleic acidsequence complementary to the sequence of miR-21; and, ii) lowering thebiological activity of miR-21 in vivo in the subject.

In another broad aspect, there is provided herein a method forincreasing chemo-sensitivity of a cancer cell in a subject comprising:i) administering to the subject a formulated anti-miR-21oligonucleotide,wherein the anti-miR-21 oligonucleotide comprising a nucleic acidsequence complementary to the sequence of miR-21, and, ii) increasingthe chemotherapy-sensitivity of the cancer cell.

In another broad aspect, there is provided herein a method comprisingadministering the composition as described herein to a subject in anamount sufficient to regulate expression of PTEN, and/or to regulate oneor more of cell proliferation, cell invasion and metastasis.

In another broad aspect, there is provided herein a method comprisingadministering the composition as described herein to a subject in anamount sufficient to regulate expression of RECK and/or TIMP, and/or toregulate one or more of cell invasion and metastasis.

In another broad aspect, there is provided herein, a pharmaceuticalcomposition comprising a composition as described claim herein, and apharmaceutically acceptable excipient.

In certain embodiments, the modified anti-miR-21 oligonucleotide isformulated with lipid nanoparticles.

In certain embodiments, wherein the nucleic acid sequence consists ofthe sequence of 5′-U*C*A*ACAUCAGUCUGAUAAG*C*U*A-3′ (SEQ ID NO:3),wherein the sequence contains phosphorothioate linkages (*).

In certain embodiments, the lipid nanoparticles comprise cationiclipids.

In certain embodiments, the cationic lipids comprise quaternary cationiclipids and tertiary cationic lipids.

In certain embodiments, the cationic liquids in the composition comprisea “QTsome™” lipid nanoparticle formulation comprised of a determinedratio of quaternary cationic lipids to tertiary cationic lipids.

In certain embodiments, the ratios of quaternary cationic lipids totertiary cationic lipids are selected from: 5:40; 15:30; 22.5:22.5;30:15; and, 40:5.

In certain embodiments, the tertiary cationic lipids comprise tertiaryamine-cationic lipids.

In certain embodiments, the tertiary amine-cationic lipids are chosenfrom DODAP, DODMA, DC-CHOL, N,N-dimethylhexadecylamine, or combinationsthereof.

In certain embodiments, the quaternary cationic lipids comprisequaternary amine-cationic lipids.

In certain embodiments, the quaternary amine-cationic lipids areselected from DOTAP, DOTMA, DDAB, or combinations thereof.

In certain embodiments, the concentration of the tertiary cationiclipids is below about 60.0 molar percent of the total lipid content.

In certain embodiments, the concentration of quaternary cationic lipidsis below about 20.0 molar percent of the total lipid content.

In certain embodiments, the composition comprises the lipids DODMA andDOTMA in a molar ratio selected from 45:0, 5:40, 15:30, 22.5:22.5,30:15, or 40:5.

In certain embodiments, the composition comprises the lipids DMHDA andDOTAP in a molar ratio selected from 90:10, 70:30, 50:50, 30:70, or10:90.

In certain embodiments, the “QTsome™” lipid nanoparticle formulation hasa formulation comprising: DOTAP/DODMA/DOPC/Cholesterol/PEG-DPPE.

In certain embodiments, the “QTsome™” lipid nanoparticle formulation hasa formulation comprising: DOTAP/DODMA/DOPC/Cholesterol/PEG-DPPE at15:25:36:20:4 mol/mol. For example, in certain embodiments, theformulation comprises: DOTAP/DODMA/DOPC/Cholesterol/PEG-DPPE, where thetotal molar percent of DOTAP and DODMA is 40:40 mol/mol.

In certain embodiments, the biological sample is a cell culture.

In certain embodiments, the cell culture is a cancer cell culture.

In certain embodiments, the cell culture is a primary cell culture froma cancer biopsy.

In certain embodiments, the biological activity is measured as theexpression levels of miR-21 target genes.

In certain embodiments, the miR-21 target genes are PTEN and RECK.

In certain embodiments, the subject has, or is suspected of having,breast cancer, ovarian cancer, lung cancer, non-small cell lungcarcinoma, squamous cell lung carcinoma, lung adenocarcinoma, large celllung carcinoma, or glioma.

In certain embodiments, the subject is a mammal.

In certain embodiments, the subject is a human.

In certain embodiments, the methods described herein further includeadministering a chemo-therapeutic agent to the subject prior to,simultaneous with, or subsequent to, administration of theanti-miR-21oligonucleotide formulation.

In certain embodiments, the chemo-therapeutic agent comprisespaclitaxel.

In certain embodiments, the composition encapsulates one or moreadditional molecules selected from nucleic acids, proteins,polysaccharides, lipids, radioactive substances, therapeutic agents,prodrugs, nutritional supplements, antibiotics, biomarkers, orcombinations thereof.

In certain embodiments, the encapsulated molecules comprise a nucleicacid selected from plasmid DNAs, antisense oligonucleotides, miRs,anti-miRs, shRNAs, siRNAs, or combinations thereof.

In certain embodiments, the encapsulation rate of therapeutic agents ornucleotides is 20% or higher.

In certain embodiments, the composition has a diameter of less than 300nm. In certain embodiments, the composition has a mean particle diameterof about 105±50 nm.

In certain embodiments, the pharmaceutical composition is administeredperorally, intravenously, subcutaneously, or transdermally. In certainembodiments, the pharmaceutical composition is prepared as an orallyadministered tablet. In certain embodiments, the pharmaceuticalcomposition is prepared as a sterile solution. In certain embodiments,the pharmaceutical composition is prepared as a sterile suspension. Incertain embodiments, the pharmaceutical composition is prepared as alyophilized powder.

In certain embodiments, the oligonucleotide agent is selected fromanti-miRs, miR mimics, antisense oligos, siRNA, aptamers, and anycombination of these classes of oligonucleotides.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIG. 1 is a representative graph showing of the upregulation of targetPTEN gene after QTsome™ encapsulating anti-miR-21 (QT/AM-21) treatmentin vitro in KB cell culture.

FIG. 2A is a graph showing miR21 expression in untreated and QT/AM-21treated cells showing down-regulation.

FIG. 2B is a graph that shows target genes (i.e., DDAH1, PTEN, RECK,PDCD$ and TIMP3) de-repression after “QTsome™” lipid nanoparticleformulation formulated anti-miR-21 (QT/AM-21) treatment in A549 lungcancer cells. The composition of “QTsome™” lipid nanoparticleformulation used was: DOTAP/DODMA/DOPC/Cholesterol/PEG-DPPE(15:25:36:20:4 mol/mol). In this composition, DOTAP is a quaternaryamine cationic lipid whereas DODMA is tertiary amine cationic lipid.

FIG. 2C is a graph showing dose dependent derepression ofmiR-21 targetsDDAH1, PTEN and RECK.

FIG. 3A and FIG. 3B are graphs showing the cell viability after thetreatment of: AM-21 (200 nM), QT, QT/AM-21 (50 nM, 100 nM, 200 nM)),paclitaxel (PTX), or the combination of PTX and QT/AM-21.

FIG. 3C is a set of photographs showing paclitaxel (PTX)chemo-sensitivity data for untreated, and cells treated with anti-miR21(AM-21) (200 nM); “QTsome™” lipid nanoparticle formulation (lipids),“QTsome™” lipid nanoparticle formulation/AM-21 (50 nM); “QTsome™” lipidnanoparticle formulation/AM-21 (100 nm); and “QTsome™” lipidnanoparticle formulation/AM-21 (200 nM).

FIG. 4A is a graph that shows target genes (i.e., PTEN, RECK and TIMP3)de-repression after “QTsome™” lipid nanoparticle formulation ofanti-miR-21 (AM21) treatment in KB cell subcutaneous xenograft.

FIG. 4B is a graph showing the measurement of tumor growth aftertreatment with “QTsome™” lipid nanoparticle formulation of anti-miR-21,showing treatment in KB cell subcutaneous xenograft.

FIG. 5A is a graph showing the tumor regression in A549 subcutaneousxenograft mouse model after QT/AM-21 treatment.

FIG. 5B is a graph showing the endpoint measurements of body weights inQT/AM-21 treated A549 subcutaneous xenograft mice.

FIG. 5C is a graph showing the endpoint measurements of liver weights inQT/AM-21 treated A549 subcutaneous xenograft mice.

FIG. 5D is a graph showing the endpoint measurements of spleen weightsin QT/AM-21 treated A549 subcutaneous xenograft mice.

FIG. 5E is a graph showing the endpoint measurements of tumor weights inQT/AM-21 treated A549 subcutaneous xenograft mice.

FIG. 5F is a graph showing the Kaplan-Meier survival, as for untreated,and QT/AM-21 (0.5 mg/kg, 1.0 mg/kg) treated xenograft mice.

FIG. 5G is a graph showing a combination therapy protocol, showing tumorvolume (mm³) for untreated, PTX, QT/AM-21 and a combination ofPTX+QT/AM-21 treated xenograft mice.

FIG. 5H is a graph showing in vivo gene regulation of DDAH1,fold-regulation (rel. GAPDH) in tumors for untreated, PTX, QT/AM-21 anda combination of PTX+QT/AM-21 treated xenograft mice.

FIG. 5I is a graph showing in vivo gene regulation of PTEN,fold-regulation (rel. GAPDH) in tumors for untreated, PTX, QT/AM-21 anda combination of PTX+QT/AM-21 treated xenograft mice.

FIG. 6A is a representative histogram that depicts the reduced percentmigration of A549 tumor cells after QT/AM-21 treatment for 48 hours.

FIG. 6B is graph showing the reduced number of invaded A549 cells in thematrigel invasion assay after QT/AM-21 treatment for 48 hours.

FIG. 7 is a schematic illustration of a “QTsome™” lipid nanoparticleformulation mechanism of action.

FIG. 8 is a graph showing: down regulation of luciferase expression inSK-HEP-1 cells. Cells expressing luciferase were treated withluciferase-specific siRNA delivered by lipid nanoparticles comprisingseveral different combinations of tertiary amine (DODMA, DMA) andquaternary amine (DOTMA, TMA), (“QTsome™” lipid nanoparticleformulation). Lipofectamine 2000 (Lipo2000) is used as a positivecontrol. Luciferase activity is expressed as a percentage relative tountreated cells.

FIG. 9 is a schematic illustration showing a design of “QTsome™” lipidnanoparticle formulation with a list of lipid nanoparticle components:

-   -   tertiary amine: 1,2-dioeyloxy-N,N-dimethyl-3-aminopropane        (DODMA);    -   quaternary amine: 1,2-dioleoyl-3-dimethylammonium-propane        (DOTAP);    -   neutral lipid: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC);    -   helper lipid: cholesterol (CHOL); and    -   PEGylating agent: N-(carbonyl-methoxypolyethyleneglycol        2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine        (DPPE-PEG).

FIG. 10A is a schematic illustration of an example of synthesis of a“QTsome™” lipid nanoparticle formulation.

FIG. 10B is a graph showing the optimization of one embodiment of aQTsome™” lipid nanoparticle formulation.

FIG. 11 is a graph showing the effect of pH on surface charge, comparingzeta potential.

FIG. 12A is a graph showing drug loading efficiency.

FIG. 12B is a graph showing particle storage stability, comparing meandiameter (nm) at: −20° C. (top line); 4° C. (bottom line); and, 25° C.(middle line) over time (days).

FIG. 13 is a graph showing QT of varying lipoamine composition preparedand evaluated for relative transfection efficiency, in fold increase inDDAH1 expression (rel. GADPH). Data represent the mean±SD of threeseparate transfections.

FIG. 14 is a graph showing stability, as measured by an in vitro releaseprofile, comparing percent drug released over time (hours).

FIG. 15 is a list of anti-miR-21 compounds 101-125 (SEQ ID NOs:4-28), inorder of appearance) which were designed and tested.

FIG. 16A is a graph showing activity of compounds 116, 118, 117, 112,113, 114, 108, 109, 110, a negative control, and anti-miR-21 of SEQ IDNO:3, in ANKRD46 (ANKRD46/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM,1.56 nM).

FIG. 16B is a graph showing activity of compounds 116, 118, 117, 112,113, 114, 108, 109, 110, a negative control, and anti-miR-21 (anti-21)of SEQ ID NO:3, in DDAH1 (DDAHUGAPDH (fold change) at 100 nM, 25 nM,6.25 nM, 1.56 nM).

FIG. 16C is a graph showing activity of compounds 116, 118, 117, 112,113, 114, 108, 109, 110, a negative control, and anti-miR-21 (anti-21)of SEQ ID NO:3, in PDCD4 (PDCD4/GAPDH (fold change) at 100 nM, 25 nM,6.25 nM, 1.56 nM).

FIG. 17A is a graph showing activity of compounds 116, 118, 117, 111,115, 119, 107, 106, 105, a negative control, and anti-miR-21 of SEQ IDNO:3 (AM-21), in DDAH1 (DDAHUGAPDH (fold change) at 100 nM, 25 nM, 6.25nM, 1.56 nM).

FIG. 17B is a graph showing activity of compounds 116, 118, 117, 111,115, 119, 107, 106, 105, a negative control, and anti-miR-21 of SEQ IDNO:3 (AM-21), in PDCD4 (PDCD4/GAPDH (fold change) at 100 nM, 25 nM, 6.25nM, 1.56 nM).

FIG. 18A is a graph showing activity of compounds 104, 103, 102, 101,120, 121, 122, 123, 124, 125, a negative control, and anti-miR-21 of SEQID NO:3 (anti-21), in ANKRD46 (ANKRD46/GAPDH (fold change) at 100 nM, 25nM, 6.25 nM, 1.56 nM).

FIG. 18B is a graph showing activity of compounds 104, 103, 102, 101,120, 121, 122, 123, 124, 125, a negative control, and anti-miR-21 of SEQID NO:3 (anti-21), in DDAH1 (DDAN1/GAPDH (fold change) at 100 nM, 25 nM,6.25 nM, 1.56 nM).

FIG. 18C is a graph showing activity of compounds 104, 103, 102, 101,120, 121, 122, 123, 124, 125, a negative control, and anti-miR-21 of SEQID NO:3 (anti-21), in PDCD4 (PDCD4/GAPDH (fold change) at 100 nM, 25 nM,6.25 nM, 1.56 nM).

FIG. 19A is a graph showing activity of compounds 114, 117, 110, 116,118, 109, 121, 101, a negative control, and anti-miR-21 of SEQ ID NO: 3(AM-21), in ANKRD46 (ANKRD46/GAPDH (fold change) at 100 nM, 25 nM, 6.25nM, 1.56 nM).

FIG. 19B is a graph showing activity of compounds 114, 117, 110, 116,118, 109, 121, 101, a negative control, and AM-21, in DDAH1 (DDAHUGAPDH(fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 19C is a graph showing activity of compounds 114, 117, 110, 116,118, 109, 121, 101, a negative control, and AM-21, in PDCD4 (PDCD4/GAPDH(fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 20A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in ANKRD46(ANKRD46/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 20B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in ANKRD46(ANKRD46/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 21A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in DDAH1(DDAH1/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 21B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in DDAH1(DDAH1/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 22A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PDCD4(PDCD4/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 22B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PDCD4(PDCD4/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 23A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PTEN(PTEN/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 23B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PTEN(PTEN/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 24A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in TIMP3(TIMP3/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 24B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in TIMP3(TIMP3/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 25A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in RECK(RECK/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 25B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in RECK(RECK/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 26A is a table showing the zeta potential without anoligonucleotide for: tertiary amine, quaternary amine, and a QTsome™lipid nanoparticle formulation.

FIG. 26B is a graph showing the zeta potential without anoligonucleotide for: tertiary amine, quaternary amine, and a QTsome™lipid nanoparticle formulation.

FIG. 27A is a table showing tumor volume (mm³) over a course of days fortreatment with saline and QT/AM-21 (2 mg/kg) in an A549 xenograft model.

FIG. 27B is a graph showing tumor volume (mm³) over a course of days forsaline and QT/AM-21 (2 mg/kg) treatment in an A549 xenograft model.

FIG. 28A is a table showing tumor volume (mm³) over a course of days forsaline and QT/AM-21 (2 mg/kg), PTX, and a combination of QT/AM-21+PTXtreatment in an A549 xenograft model.

FIG. 28B is a graph showing tumor volume (mm³) over a course of days forsaline and QT/AM-21 (2 mg/kg), PTX, and a combination of QT/AM-21+PTXtreatment in an A549 xenograft model.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments are described herein in the context of lipidnanoparticles. Those of ordinary skill in the art will realize that thefollowing detailed description of the embodiments is illustrative onlyand not intended to be in any way limiting. Other embodiments willreadily suggest themselves to such skilled persons having the benefit ofthis disclosure. Reference to an “embodiment,” “aspect,” or “example”herein indicate that the embodiments of the invention so described mayinclude a particular feature, structure, or characteristic, but notevery embodiment necessarily includes the particular feature, structure,or characteristic. Further, repeated use of the phrase “in oneembodiment” does not necessarily refer to the same embodiment, althoughit may.

Not all of the routine features of the implementations or processesdescribed herein are shown and described. It will, of course, beappreciated that in the development of any such actual implementation,numerous implementation-specific decisions will be made in order toachieve the developer's specific goals, such as compliance withapplication- and business-related constraints, and that these specificgoals will vary from one implementation to another and from onedeveloper to another. Moreover, it will be appreciated that such adevelopment effort might be complex and time-consuming, but wouldnevertheless be a routine undertaking for those of ordinary skill in theart having the benefit of this disclosure.

General Description and Definitions

Lung cancer is the most common cancer worldwide. The 5-year relativesurvival rate about 15.7%. Non-small cell lung cancer (NSCLC) accountsfor the majority of lung cancers, and is generally categorized into thefollowing types: squamous cell carcinoma, adenocarcinoma, and large cellcarcinoma. The risk factors include exposure to cigarette smoke or otherairborne carcinogens. NSCLC originates in epithelial cells of thecentral bronchiole and terminal alveoli. NSCLC is relatively insensitiveto chemotherapy and radiation therapy. miR-21 is upregulated in NSCLC,and while not wishing to be bound by theory, the inventors herein nowbelieve that such upregulated miR-21 is target for treatment of NSCLC.

microRNA (miRs) miRs are often differentially expressed between tumorand non-tumor tissue. miRNAs are Small non-protein coding RNA, generally20-25 nucleotides long, and are capable of regulating hundreds of mRNAsin multiple biological pathways. miRNAs are incorporated in toRNA-induced silencing complexes (RISC) and regulate gene expressionthrough RNA interference. Recognition by RISC leads to target mRNAtranslational repression. In addition, matching of targets generallyoccurs in the 3′-UTR of mRNA.

miR-21 is a regulator for a large number of genes. Targets includepathways responsible for cell proliferation, invasion, metastasis, andevasion of apoptosis. Administration of an oligonucleotide miR-inhibitoragainst miR-21 (anti-miR-21, AM-21) is now shown herein to reversemiR-21 activity.

The genes PTEN and PDCD4 function as tumor suppressors. PTEN inhibitsAkt pathway via reversal of PI3K phosphorylation, while PDCD4 inhibitstranslation initiation factors eIF4A and eIF4G. miR-21 expression isinversely correlated with PTEN and PDCD4.

Asymmetric dimethylarginine (ADMA) is a competitive inhibitor ofendothelial nitric oxide synthase. Nitric oxide is responsible forinhibition of apoptosis and regulation of angiogenesis.Dimethylaminohydrolase (DDAH1) is responsible for ADMA elimination andis correlated with increased miR-21 expression.

Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) andmetalloproteinase inhibitor 3 (TIMP3) function as metalloproteinaseinhibitors. RECK and TIMP3 regulate tumor cell invasion, metastasis, andangiogenesis.

Paclitaxel (PTX) is a common treatment in lung, ovarian, and breastcancers. Several studies suggest that resistance to PTX is linked tooverexpression of miR-21 via hypoxia-inducible factor-1α (HIF-1α).

However there are barriers to using miRNAs and anti-miRNAs due, in part,to the difficulty in the delivery of the miRNAs/anti-miRNAs to a targetsite. In general, the characteristics of miRNAs/anti-miRNAs themselvespose difficulties as the miRNAs/anti-miRNAs are: high molecular weight,have high anionic charge, generally show instability in serum, there israpid clearance, they have poor pharmacokinetic properties, and maycause undesirable off-target effects.

Subject” means a human or non-human animal selected for treatment ortherapy.

“Subject in need thereof” means a subject identified as in need of atherapy or treatment. In certain embodiments, a subject has livercancer. In such embodiments, a subject has one or more clinicalindications of liver cancer or is at risk for developing liver cancer.

“At risk for developing cancer” means the state in which a subject ispredisposed to developing cancer.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration,” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, orintramuscular administration.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Chemoembolization” means a procedure in which the blood supply to atumor is blocked surgically, mechanically, or chemically andchemotherapeutic agents are administered directly into the tumor.

“Duration” means the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent orpharmaceutical composition are administered.

“Therapy” means a disease treatment method. In certain embodiments,therapy includes, but is not limited to, chemotherapy, surgicalresection, liver transplant, and/or chemoembolization.

“Treatment” means the application of one or more specific proceduresused for the cure or amelioration of a disease. In certain embodiments,the specific procedure is the administration of one or morepharmaceutical agents.

“Amelioration” means a lessening of severity of at least one indicatorof a condition or disease. In certain embodiments, amelioration includesa delay or slowing in the progression of one or more indicators of acondition or disease. The severity of indicators may be determined bysubjective or objective measures which are known to those skilled in theart.

“Prevention” refers to delaying or forestalling the onset or developmentor progression of a condition or disease for a period of time, includingweeks, months, or years.

“Prevent the onset of” means to prevent the development a condition ordisease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Delay the onset of” means to delay the development of a condition ordisease in a subject who is at risk for developing the disease orcondition. In certain embodiments, a subject at risk for developing thedisease or condition receives treatment similar to the treatmentreceived by a subject who already has the disease or condition.

“Therapeutic agent” means a pharmaceutical agent used for the cure,amelioration or prevention of a disease.

“Anti-cancer therapy” means a therapy aimed at treating or preventingcancer. In certain embodiments, anti-cancer therapy compriseschemotherapy. In certain embodiments, anti-cancer therapy comprisesradiation therapy.

“Chemotherapeutic agent” means a pharmaceutical agent used to treatcancer.

“Chemotherapy” means treatment of a subject with one or morepharmaceutical agents for the treatment of cancer.

“Metastasis” means the process by which cancer spreads from the place atwhich it first arose as a primary tumor to other locations in the body.The metastatic progression of a primary tumor reflects multiple stages,including dissociation from neighboring primary tumor cells, survival inthe circulation, and growth in a secondary location.

“Overall survival time” means the time period for which a subjectsurvives after diagnosis of or treatment for a disease. In certainembodiments, the disease is cancer.

“Progression-free survival” means the time period for which a subjecthaving a disease survives, without the disease getting worse. In certainembodiments, progression-free survival is assessed by staging or scoringthe disease. In certain embodiments, progression-free survival of asubject having liver cancer is assessed by evaluating tumor size, tumornumber, and/or metastasis.

“Biomarker” means a substance that is used as an indicator of a biologicstate. Biomarkers are objectively measured and evaluated as an indicatorof normal biologic processes, pathogenic processes, or pharmacologicresponses to a therapeutic intervention.

“Cancer biomarker” means a substance that is used as an indicator of acancerous state. For example, a cancer biomarker may indicate thepresence of cancer, or the response to an anti-cancer therapy.

“Improved organ function” means the change in organ function towardnormal organ function. In certain embodiments, organ function isassessed by measuring molecules found in a subject's blood.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration. In certain embodiments, a dose may beadministered in two or more boluses, tablets, or injections.

“Dosage unit” means a form in which a pharmaceutical agent is provided.

“Therapeutically effective amount” refers to an amount of apharmaceutical agent that provides a therapeutic benefit to an animal.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual that includes a pharmaceutical agent.

“Pharmaceutical agent” means a substance that provides a therapeuticeffect when administered to a subject.

“Active pharmaceutical ingredient” means the substance in apharmaceutical composition that provides a desired effect.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean any nucleic acid capable of beingtargeted by antisense compounds.

“Targeting” means the process of design and selection of nucleobasesequence that will hybridize to a target nucleic acid and induce adesired effect.

“Targeted to” means having a nucleobase sequence that will allowhybridization to a target nucleic acid to induce a desired effect. Incertain embodiments, a desired effect is reduction of a target nucleicacid.

“Expression” means any functions and steps by which a gene's codedinformation is converted into structures present and operating in acell.

“Natural nucleobase” means a nucleobase that is unmodified relative toits naturally occurring form.

A “stabilizing modification” means a modification to a nucleoside thatprovides enhanced stability to a modified oligonucleotide, in thepresence of nucleases, relative to that provided by nucleosides or2′-deoxynucleosides linked by phosphodiester internucleoside linkages.For example, in certain embodiments, a stabilizing modification is astabilizing nucleoside modification.

Compositions of the present invention comprise oligomeric compoundscomprising oligonucleotides having nucleobase sequences that shareidentity with endogenous miRNA or miRNA precursor nucleobase sequences.An oligonucleotide selected for inclusion in a composition of thepresent invention may be one of a number of lengths. Such anoligonucleotide can be from 7 to 100 linked nucleosides in length. Forexample, an oligonucleotide sharing nucleobase identity with a miRNA maybe from 7 to 30 linked nucleosides in length. An oligonucleotide sharingidentity with a miRNA precursor may be up to 100 linked nucleosides inlength.

In certain embodiments, an oligonucleotide consists of 7 to 30 linkednucleosides. In certain embodiments, an oligonucleotide consists of 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 28, 29, or 30 linked nucleotides.

In certain embodiments, an oligonucleotide consists of 15 to 30 linkednucleosides. In certain embodiments, an oligonucleotide consists of 15linked nucleosides. In certain embodiments, an oligonucleotide consistsof 16 linked nucleosides. In certain embodiments, an oligonucleotideconsists of 17 linked nucleosides. In certain embodiments, anoligonucleotide consists of 18 linked nucleosides. In certainembodiments, an oligonucleotide consists of 19 linked nucleosides. Incertain embodiments, an oligonucleotide consists of 20 linkednucleosides. In certain embodiments, an oligonucleotide consists of 21linked nucleosides. In certain embodiments, an oligonucleotide consistsof 22 linked nucleosides. In certain embodiments, an oligonucleotideconsists of 23 linked nucleosides. In certain embodiments, anoligonucleotide consists of 24 linked nucleosides. In certainembodiments, an oligonucleotide consists of 25 linked nucleosides. Incertain embodiments, an oligonucleotide consists of 26 linkednucleosides. In certain embodiments, an oligonucleotide consists of 27linked nucleosides. In certain embodiments, an oligonucleotide consistsof 28 linked nucleosides. In certain embodiments, an oligonucleotideconsists of 29 linked nucleosides. In certain embodiments, anoligonucleotide consists of 30 linked nucleosides.

In certain embodiments, an oligonucleotide consists of 19 to 23 linkednucleosides. In certain embodiments, an oligonucleotide is from 40 up to50, 60, 70, 80, 90, or 100 linked nucleosides in length.

In certain embodiments, an oligonucleotide has a sequence that has acertain identity to a miRNA or a precursor thereof. Nucleobase sequencesof mature miRNAs and their corresponding stem-loop sequences describedherein are the sequences found in miRBase, an online searchable databaseof miRNA sequences and annotation, found at the websitemicroRNA“dot”sanger“dot”ac“dot”uk. Entries in the miRBase Sequencedatabase represent a predicted hairpin portion of a miRNA transcript(the stem-loop), with information on the location and sequence of themature miRNA sequence. The miRNA stem-loop sequences in the database arenot strictly precursor miRNAs (pre-miRNAs), and may in some instancesinclude the pre-miRNA and some flanking sequence from the presumedprimary transcript. The miRNA nucleobase sequences described hereinencompass any version of the miRNA, including the sequences described inRelease 10.0 of the miRBase sequence database and sequences described inany earlier Release of the miRBase sequence database. A sequencedatabase release may result in the re-naming of certain miRNAs. Asequence database release may result in a variation of a mature miRNAsequence. The compositions of the present invention encompass oligomericcompound comprising oligonucleotides having a certain identity to anynucleobase sequence version of a miRNAs described herein.

In certain embodiments, an oligonucleotide has a nucleobase sequence atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% Aidentical to the miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleobases. Accordingly, in certain embodiments the nucleobase sequenceof an oligonucleotide may have one or more non-identical nucleobaseswith respect to the miRNA. In certain embodiments, the miRNA is miR-21.

In certain embodiments, an oligonucleotide has a nucleobase sequence atleast 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% Aidentical to the precursor over a region of 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleobases. Incertain embodiments, the miRNA precursor is a miR-21 precursor.

Compositions of the present invention may comprise oligonucleotideshaving a percentage region identity and percentage overall identity thatare different from one another. In certain embodiments, a region of thenucleobase sequence of an oligonucleotide is 100% identical to thenucleobase sequence of the miRNA, but the oligonucleotide does not have100% overall identity to the entire miRNA. In certain such embodiments,the number of nucleosides of the oligonucleotide is greater than thelength of the miRNA, but the oligonucleotide has a region that is 100%identical to the miRNA.

Compositions described herein may comprise oligonucleotides having seedregion identity with a miRNA. In certain embodiments, the nucleobasesequence of an oligonucleotide has at least 80% seed region identitywith the nucleobase sequence of a miRNA. In certain embodiments, thenucleobase sequence of an oligonucleotide has at least 85% seed regionidentity with the nucleobase sequence of a miRNA. In certainembodiments, the nucleobase sequence of an oligonucleotide has at least90% seed region identity with the nucleobase sequence of a miRNA. Incertain embodiments, the nucleobase sequence of an oligonucleotide hasat least 95% seed region identity with the nucleobase sequence of amiRNA. In certain embodiments, the nucleobase sequence of anoligonucleotide has 100% seed region identity with the nucleobasesequence of a miRNA.

Compositions herein may also comprise locked nucleic acid (LNA)substituted oligonucleotides. Locked nucleic acid substitutedoligonucleotides have increased nuclease stability and greatly increasedRNA target binding affinity. Therefore, locked nucleic acid is useful toenhance the activity of anti-miRs. For example, the compounds 101-119shown in FIG. 15 (SEQ ID. NOs:3-21) are LNA-containing oligonucleotides.In other, non-limiting examples, other types of modifications such asunlocked nucleic acid (UNA), morpholine substituted, 2′-methoxyethyl(MOE), 2′-F, peptide nucleic acid (PNA), conformationally restrictednucleosides (CRN) are also useful to construct anti-miRs.

Certain Nanoparicles

The nanoparticles described herein are especially useful for thedelivery of miRNAs/anti-miRNAs. The nanoparticles include cationicpolymers or lipid components which provide improved pharmacokinetics andincreased cellular uptake. The nanoparticles described herein provideprotection of the anti-miR against degradation. Also, the nanoparticlesprovide improved safety parameters, provide passive targeting byenhanced permeation and retention (EPR) effect, and the capability oftargeting specific cell types with ligands.

Endosomal escape is a rate limiting factor in anti-miR delivery.Described herein are fusogenic lipids that are used to help thenanoparticle avoid degradation in the lysosome by promoting endosomallysis. Useful fusogenic lipids include phosphatidylethanolamines andcholesterol.

The lipid nanoparticles described herein provide a useful platform forthe delivery of both traditional therapeutic compounds and NA-basedtherapies. Drugs formulated using lipid nanoparticles provide desirablepharmacokinetic (PK) properties in vivo, such as increased bloodcirculation time and increased accumulation at the site of solid tumorsdue to enhanced permeability and retention (EPR) effect. Moreover, incertain embodiments, the lipid nanoparticles may be surface-coated withpolyethylene glycol to reduce opsonization of lipid nanoparticles byserum proteins and the resulting RES-mediated uptake, and/or coated withcell-specific ligands to provide targeted drug delivery.

It is desired that the zeta potential of lipid nanoparticles not beexcessively positive or negative for systemic delivery. Lipidnanoparticles with a highly positive charge tend to interactnon-specifically with non-target cells, tissues, and circulating plasmaproteins, and may cause toxicity and rapid clearance. Alternatively,lipid nanoparticles with a highly negative charge cannot effectivelyincorporate NAs, which are themselves negatively charged, and maytrigger rapid RES-mediated clearance, reducing therapeutic efficacy.Lipid nanoparticles with a neutral to moderate charge are best suitedfor in vivo drug and gene delivery.

Provided herein are lipid nanoparticles (lipid nanoparticles) withimproved transfection activity. The lipid nanoparticles may eitherpartition hydrophobic molecules within the lipid membrane or encapsulatewater-soluble particles or molecules within the aqueous core.

In certain embodiments, the lipid nanoparticles are produced bycombining cationic lipids with quaternary amine headgroups and cationiclipids with tertiary amine headgroups. In certain embodiment, the lipidnanoparticles are small peptidic lipid nanoparticles (SPLN) and comprisea peptide such as gramicidin or JTS1. Exemplary lipid nanoparticles aredisclosed in PCT WO2013/177419, the entire contents of which areexpressly incorporated herein.

Combinations of these different embodiments are further provided. Thelipid nanoparticles have a diameter of less than 300 nm, or inparticular embodiments between about 50 and about 200 nm. These lipidnanoparticles show enhanced transfection and reduced cytotoxicity,especially under high serum conditions found during systemicadministration. The lipid nanoparticles are applicable to a wide rangeof current therapeutic agents and systems, serum stability, and targeteddelivery, with high transfection efficiency.

The term “lipid nanoparticle” as used herein refers to any vesiclesformed by one or more lipid components. The lipid nanoparticleformulations described herein may include cationic lipids. Cationiclipids are lipids that carry a net positive charge. The positive chargeis used for association with negatively charged therapeutics such asASOs and anti-miRs via electrostatic interaction. In specificembodiments and in abbreviations in the Figures, the lipid nanoparticlemay be referred to herein as “QTsome™ lipid nanoparticle formulation,”as “QTsome™” or as “QT.”

Suitable cationic lipids include, but are not limited to:3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol hydrochloride(DC-Chol); 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP);1,2-dioleoyl-3-dimethylammonium-propane (DODAP);dimethyldioctadecylammonium bromide salt (DDAB);1,2-dilauroyl-sn-glycero-3-ethylphosphocholine chloride (DL-EPC);N-(1-(2,3-dioleyloyx) propyl)-N-N-N-trimethyl ammonium chloride (DOTMA);N-(1-(2,3-dioleyloyx) propyl)-N-N-N-dimethyl ammonium chloride (DODMA);N,N-dioctadecyl-N,N-dimethylammonium chloride (DODAC);N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA); 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); dioctadecylamidoglycylspermine (DOGS); neutrallipids conjugated to cationic modifying groups; and combinationsthereof. In addition, a number of cationic lipids in availablepreparations could be used, such as LIPOFECTIN® (from GIBCO/BRL),LIPOFECTAMINE® (from GIBCO/MRL), siPORT NEOFX® (from AppliedBiosystems), TRANSFECTAM® (from Promega), and TRANSFECTIN® (from Bio-RadLaboratories, Inc.). The cationic lipids of the present disclosure maybe present at concentrations ranging from about 0 to about 80.0 molarpercent of the lipids in the formulation, or from about 5.0 to about50.0 molar percent of the formulation.

In certain embodiments, the lipid nanoparticle formulations presentlydisclosed may also include anionic lipids. Anionic lipids are lipidsthat carry a net negative charge at physiological pH. These anioniclipids, when combined with cationic lipids, are useful to reduce theoverall surface charge of lipid nanoparticles and introduce pH-dependentdisruption of the lipid nanoparticle bilayer structure, facilitatingnucleotide release by inducing nonlamellar phases at acidic pH or inducefusion with the cellular membrane.

Examples of suitable anionic lipids include, but are not limited to:fatty acids such as oleic, linoleic, and linolenic acids; cholesterylhemisuccinate;1,2-di-O-tetradecyl-sn-glycero-3-phospho-(1′-rac-glycerol) (Diether PG);1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt);1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt);1-hexadecanoyl,2-(9Z,12Z)-octadecadienoyl-sn-glycero-3-phosphate;1,2-dioleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DOPG);dioleoylphosphatidic acid (DOPA); and1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS); anionic modifyinggroups conjugated to neutral lipids; and combinations thereof. Theanionic lipids of the present disclosure are present at concentrationsup to about 60.0 molar percent of the formulation, or from about 5.0 toabout 25.0 molar percent of the formulation.

In certain embodiments, charged lipid nanoparticles are advantageous fortransfection, but off-target effects such as cytotoxicity andRES-mediated uptake may occur. Hydrophilic molecules such aspolyethylene glycol (PEG) may be conjugated to a lipid anchor andincluded in the lipid nanoparticles described herein to discourage lipidnanoparticle aggregation or interaction with membranes. Hydrophilicpolymers may be covalently bonded to lipid components or conjugatedusing crosslinking agents to functional groups such as amines.

Suitable conjugates of hydrophilic polymers include, but are not limitedto: polyvinyl alcohol (PVA); polysorbate 80;1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000(DSPE-PEG2000); D-alpha-tocopheryl polyethylene glycol 1000 succinate(TPGS); dimyristoylphosphatidylethanolamine-PEG2000 (DMPE-PEG2000); anddip almitoylphosphatidlyethanolamine-PEG2000 (DPPE-PEG2000). Thehydrophilic polymer may be present at concentrations ranging from about0 to about 15.0 molar percent of the formulation, or from about 5.0 toabout 10.0 molar percent of the formulation. The molecular weight of thePEG used is between about 100 and about 10,000 Da, or from about 100 toabout 2,000 Da.

The lipid nanoparticles described herein may further comprise neutraland/or amphipathic lipids as helper lipids. These lipids are used tostabilize the formulation, reduce elimination in vivo, or increasetransfection efficiency. The lipid nanoparticles may be formulated in asolution of saccharides such as, but not limited to, glucose, sorbitol,sucrose, maltose, trehalose, lactose, cellubiose, raffinose,maltotriose, dextran, or combinations thereof, to promote lyostabilityand cryostability.

Neutral lipids have zero net charge at physiological pH. One or acombination of several neutral lipids may be included in any lipidnanoparticle formulation disclosed herein.

Suitable neutral lipids include, but are not limited to:phosphatidylcholine (PC), phosphatidylethanolamine, ceramide,cerebrosides, sphingomyelin, cephalin, cholesterol, diacylglycerols,glycosylated diacylglycerols, prenols, lysosomal PLA2 substrates,N-acylglycines, and combinations thereof.

Other suitable lipids include, but are not limited to:phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine,phosphatidylglycerol, phosphatidylcholine, andlysophosphatidylethanolamine; sterols such as cholesterol, demosterol,sitosterol, zymosterol, diosgenin, lanostenol, stigmasterol,lathosterol, and dehydroepiandrosterone; and sphingolipids such assphingosines, ceramides, sphingomyelin, gangliosides,glycosphingolipids, phosphosphingolipids, phytoshingosine; andcombinations thereof.

The lipid nanoparticle formulations described herein may furthercomprise fusogenic lipids or fusogenic coatings to promote membranefusion. Examples of suitable fusogenic lipids include, but are notlimited to, glyceryl mono-oleate, oleic acid, palmitoleic acid,phosphatidic acid, phosphoinositol 4,5-bisphosphate (PIP₂), andcombinations thereof.

The lipid nanoparticle formulations described herein may furthercomprise cationic lipids. The headgroups of such lipids may be primary,secondary, tertiary, or quaternary amines in nature. In certainembodiments, the lipid nanoparticles comprise a mixture of tertiary andquaternary amines.

Suitable tertiary aminolipids include, but are not limited to: DODAP;DODMA; N,N-dimethylhexadecylamine (DMHDA); DC-CHOL;1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-distearyloxy-N,N-Dimethylaminopropane (DSDMA), ionizable cationiclipids DLinDAP, DLinKDMA, and DLinKC2-DMA.

Suitable quaternary aminolipids include, but are not limited to: DOTAP,DOTMA, DDAB, 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt(DLin-DMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt(DLin-TAP.Cl), 1,2-Dilinolenyloxy-3-trimethylaminopropane chloride salt.(DLen-TMA.Cl), and1,2-Dilinolenyloxy-3-trimethylaminopropane chloridesalt (DLen-TAP.Cl).

Combinations of multiple aminolipids, particularly of tertiary andquaternary cationic lipids, are beneficial towards lipid nanoparticledelivery of therapeutic agents. In certain embodiments, the cationiclipids may be present in concentrations up to about 60 molar percentcombined.

The lipid nanoparticle formulations described here may further comprisecationic polymers or conjugates of cationic polymers. Cationic polymersor conjugates thereof may be used alone or in combination with lipidnanocarriers.

Suitable cationic polymers include, but are not limited to:polyethylenimine (PEI); pentaethylenehexamine (PEHA); spermine;spermidine; poly(L-lysine); poly(amido amine) (PAMAM) dendrimers;polypropyleneiminie dendrimers; poly(2-dimethylamino ethyl)-methacrylate(pDMAEMA); chitosan; tris(2-aminoethyl)amine and its methylatedderivatives; and combinations thereof. Chain length and branching areimportant considerations for the implementation of polymeric deliverysystems. High molecular weight polymers such as PEI (MW 25,000) are usedas transfection agents, but suffer from cytotoxicity. Low molecularweight PEI (MW 600) does not cause cytotoxicity, but is limited due toits inability to facilitate stable condensation with nucleic acids.

Anionic polymers may be incorporated into the lipid nanoparticleformulations presently disclosed as well. Suitable anionic polymersinclude, but are not limited to: poly(propylacrylic acid) (PPAA);poly(glutamic acid) (PGA); alginates; dextrans; xanthans; derivatizedpolymers; and combinations thereof.

In certain embodiments, the lipid nanoparticle formulation includesconjugates of polymers. The conjugates may be crosslinked to targetingagents, lipophilic moieties, peptides, proteins, or other molecules thatincrease the overall therapeutic efficacy.

Suitable crosslinking agents include, but are not limited to:N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP); dimethyl3,3′-dithiobispropionimidate (DTBP); dicyclohexylcarbodiimide (DCC);diisopropyl carbodiimide (DIC);1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC);N-hydroxysulfosuccinimide (Sulfo-NHS); N′-N′-carbonyldiimidazole (CDI);N-ethyl-5-phenylisoxazolium-3′sulfonate (Woodward's reagent K); andcombinations thereof.

The lipid nanoparticle formulations may further comprise peptides and/orproteins. Peptides and proteins, especially those derived from bacteriaand viruses or used as antibiotic agents, may aid in membranepermeation. The peptides or proteins may be directly mixed with lipids,covalently attached, or conjugated to lipid moieties with crosslinkingagents.

Suitable peptides and proteins include, but are not limited to:gramicidin A, B, C, D, and S; HA2; JTS-1; proteinase K (PrK);trichorovin-Xlla (TV-Xlla); rabies virus glycoprotein (RVG);interleukin-β; HIV-Tat; herpes simplex virus (HSV) VP22 protein; andcombinations thereof. In certain embodiments, JTS-1 and/or gramicidin isused at about 0 to about 40 molar percent. In certain embodiments, PrKat a concentration of about 0 to about 30 molar percent is applied bydirect mixing with oligonucleotide or conjugation to hexadecylisothiocyanate for lipid nanoparticle surface coating of PrK.

The addition of targeting agents to the lipid nanoparticle providesincreased efficacy over passive targeting approaches. Targeting involvesincorporation of specific targeting moieties such as, but not limitedto, ligands or antibodies against cell surface receptors, peptides,lipoproteins, glycoproteins, hormones, vitamins, antibodies, antibodyfragments, prodrugs, and conjugates or combinations of these moieties.

In certain embodiments, maximization of targeting efficiency includesthe surface coating of the lipid nanoparticle with the appropriatetargeting moiety rather than encapsulation of the targeting agent. Thismethod optimizes interaction with cell surface receptors.

It is to be understood that targeting agents may be either directlyincorporated into the lipid nanoparticle during synthesis or added in asubsequent step. Functional groups on the targeting moiety as well asspecifications of the therapeutic application (e.g., degradable linkage)dictate the appropriate means of incorporation into the lipidnanoparticle. Targeting moieties that do not have lipophilic regionscannot insert into the lipid bilayer of the lipid nanoparticle directlyand require prior conjugation to lipids before insertion or must form anelectrostatic complex with the lipid nanoparticles.

Also, under certain circumstances, a targeting ligand cannot directlybind to a lipophilic anchor. In these circumstances, a molecular bridgein the form of a crosslinking agent may be utilized to facilitate theinteraction. In certain embodiments, it is advantageous to use acrosslinking agent if steric restrictions of the anchored targetingmoiety prevent sufficient interaction with the intended physiologicaltarget. Additionally, if the targeting moiety is only functional undercertain orientations (e.g., monoclonal antibody), linking to a lipidanchor via crosslinking agent is beneficial. Traditional methods ofbioconjugation may be used to link targeting agents to lipidnanoparticles. Reducible or hydrolysable linkages may be applied toprevent accumulation of the formulation in vivo and subsequentcytotoxicity.

Various methods of lipid nanoparticle preparation are suitable tosynthesize the lipid nanoparticles of the present disclosure. Forexample, ethanol dilution, freeze-thaw, thin film hydration, sonication,extrusion, high pressure homogenization, detergent dialysis,microfluidization, tangential flow diafiltration, sterile filtration,and/or lyophilization may be utilized. Additionally, several methods maybe employed to decrease the size of the lipid nanoparticles. Forexample, homogenization may be conducted on any devices suitable forlipid homogenization such as an Avestin Emulsiflex C5® device. Extrusionmay be conducted on a Lipex Biomembrane extruder using a polycarbonatemembrane of appropriate pore size (0.05 to 0.2 μm). Multiple particlesize reduction cycles may be conducted to minimize size variation withinthe sample. The resultant lipid nanoparticles may then be passed througha size exclusion column such as Sepharose CL-4B or processed bytangential flow diafiltration to purify the lipid nanoparticles.

Any embodiment of the lipid nanoparticles described herein may furtherinclude ethanol in the preparation process. The incorporation of about30-50% ethanol in lipid nanoparticle formulations destabilizes the lipidbilayer and promotes electrostatic interactions among charged moietiessuch as cationic lipids with anionic oligonucleotides, such as ASO andsiRNA. Lipid nanoparticles prepared in high ethanol solution are dilutedbefore administration. Alternatively, ethanol may be removed bydialysis, or diafiltration, which also removes non-encapsulated NA.

In certain embodiment, it is desirable that the lipid nanoparticles besterilized. This may be achieved by passing of the lipid nanoparticlesthrough a 0.2 or 0.22 μm sterile filter with or without pre-filtration.

Physical characterization of the lipid nanoparticles can be carriedthrough many methods. Dynamic light scattering (DLS) or atomic forcemicroscopy (AFM) can be used to determine the average diameter and itsstandard deviation. In certain embodiments, it is especially desirablethat the lipid nanoparticles have about a 200 nm diameter. Zetapotential measurement via zeta potentiometer is useful in determiningthe relative stability of particles. Both dynamic light scatteringanalysis and zeta potential analysis may be conducted with dilutedsamples in deionized water or appropriate buffer solution. Cryogenictransmission electron microscopy (Cryo-TEM) and scanning electronmicroscopy (SEM) may be used to determine the detailed morphology oflipid nanoparticles.

The lipid nanoparticles described herein are stable under refrigerationfor several months. Lipid nanoparticles requiring extended periods oftime between synthesis and administration may be lyophilized usingstandard procedures. A cryoprotectant such as 10% sucrose may be addedto the lipid nanoparticle suspension prior to freezing to maintain theintegrity of the formulation. Freeze drying loaded lipid nanoparticleformulations is recommended for long term stability.

Quaternary Amine-Cationic and Tertiary Amine-Cationic Lipid NanoparticleFormulations (“QTSOME™”)

While the physical characteristics of lipid nanoparticles promoteenhanced permeation and retention (EPR) in the fenestrated tumorvasculature, endosomal escape remains a challenge for conventional lipidnanoparticle formulations. To this end, lipid nanoparticles comprisingpositively charged quaternary or tertiary amine-based cationic lipidsfor the complexation of nucleic acids have been developed. Quaternaryamine-based cationic lipids carry a permanent positive charge and arecapable of forming stable electrostatic complexes with nucleic acids.Tertiary amine-cationic lipids, however, are conditionally ionizable andtheir positive charge is largely regulated by pH. Provided herein arelipid nanoparticles comprising a combination of quaternary and tertiaryamine-cationic lipids (”QTsome™” lipid nanoparticle formulations), whichprovides a mechanism by which therapeutic agents may be released fromlipid nanoparticles within the endosome. “QTsome™” lipid nanoparticleformulations are conditionally ionizable and facilitate disruption ofthe lipid bilayer and oligonucleotide endosomal release under the acidicconditions of the endosome. Quaternary amino-cationic lipids arepermanently charged, ensuring strong interaction between the lipids andthe oligonucleotide, thereby ensuring stability. The combination oftertiary and quaternary cationic lipids provides an optimum pH responseprofile that is not possible with each class of lipid individually.“QTsome™” lipid nanoparticle formulations are more active than regularcationic liposomes in transfecting cells.

“QTsome™” lipid nanoparticle formulations demonstrate greatertransfection activity than standard cationic lipid formulations. Finetuning the balance between quaternary and tertiary amine-cationic lipidsallows for the precise controlled release of nucleic acids into thecytosol. In a particular embodiment, the use of particular releaseparameters provides a technique whereby the activity of nucleicacid-based therapeutics can be maximized For example, it is noted thattertiary amine-cationic lipids have pH-dependent ionization profileswhen used alone. Since a single lipid species may not provide a desiredlevel of control of lipid nanoparticle charge characteristics, acombination of a tertiary and a quaternary amine-cationic lipid can beused, thus resulting in significantly improved activity of suchcombinations in siRNA delivery.

It is to be understood that both the tumor environment and endosomeshave acidic pH. While not wishing to be bound by theory, it is nowbelieve that when QTsomes get into the tumor, they may acquire positivesurface charge due to the acidity of the tumor environment and interactwith the plasma membrane of tumor cells. The endosomes are even moreacidic and can trigger further increase in cationic charge; but, bothtypes of pH trigger contribute to the in vivo activity of QTsomes.

Applications

Depending on the application, the lipid nanoparticles disclosed hereinmay be designed to favor characteristics such as increased interactionwith nucleic acids, increased serum stability, lower RES-mediateduptake, targeted delivery, or pH sensitive release within the endosome.Because of the varied nature of lipid nanoparticle formulations, any oneof the several methods provided herein may be applied to achieve aparticular therapeutic aim. Cationic lipids, anionic lipids, PEG-lipids,neutral lipids, fusogenic lipids, aminolipids, cationic polymers,anionic polymers, polymer conjugates, peptides, targeting moieties, andcombinations thereof may be applied to meet specific aims.

The lipid nanoparticles described herein can be used as platforms fortherapeutic delivery of oligonucleotide (ON) therapeutics, such assiRNA, shRNA, miRNA, anti-miR, and antisense ODN. These therapeutics areuseful to manage a wide variety of diseases such as various types ofcancers, leukemias, viral infections, and other diseases. For instance,targeting moieties such as cyclic-RGD, folate, transferrin, orantibodies greatly enhance activity by enabling targeted drug delivery.A number of tumors overexpress receptors on their cell surface.Non-limiting examples of suitable targeting moieties include transferrin(Tf), folate, low density lipoprotein (LDL), and epidermal growthfactors. In addition, tumor vascular endothelium markers such asalpha-v-beta-3 integrin and prostate-specific membrane antigen (PSMA)are valuable as targets for targeted lipid nanoparticles. In certainembodiments, lipid nanoparticle formulations having particles measuringabout 300 nm or less in diameter with a zeta potential of less than 50mV and an encapsulation efficiency of greater than 20.0% are useful forNA delivery.

Implementation of embodiments of the lipid nanoparticle formulationsdescribed herein alone or in combination with one another synergizeswith current paradigms of lipid nanoparticle design.

A wide spectrum of therapeutic agents may be used in conjunction withthe lipid nanoparticles described herein. Non-limiting examples of suchtherapeutic agents include antineoplastic agents, anti-infective agents,local anesthetics, anti-allergics, antianemics, angiogenesis,inhibitors, beta-adrenergic blockers, calcium channel antagonists,anti-hypertensive agents, anti-depressants, anti-convulsants,anti-bacterial, anti-fungal, anti-viral, anti-rheumatics,anthelminithics, antiparasitic agents, corticosteroids, hormones,hormone antagonists, immunomodulators, neurotransmitter antagonists,anti-diabetic agents, anti-epileptics, anti-hemmorhagics,anti-hypertonics, antiglaucoma agents, immunomodulatory cytokines,sedatives, chemokines, vitamins, toxins, narcotics, imaging agents, andcombinations thereof.

Nucleic acid-based therapeutic agents are highly applicable to the lipidnanoparticle formulations of the present disclosure. Examples of suchnucleic acid-based therapeutic agents include, but are not limited to:pDNA, siRNA, miRNA, anti-miRNA, ASO, and combinations thereof. Toprotect from serum nucleases and to stabilize the therapeutic agent,modifications to the substituent nucleic acids and/or phosphodiesterlinker can be made. Such modifications include, but are not limited to:backbone modifications (e.g., phosphothioate linkages); 2′ modifications(e.g., 2′-O-methyl substituted bases); zwitterionic modifications(6′-aminohexy modified ODNs); the addition of a lipophilic moiety (e.g.,fatty acids, cholesterol, or cholesterol derivatives); and combinationsthereof. The modified sequences synergize with the lipid nanoparticleformulations disclosed herein. For example, addition of a 3′-cholesterolto an ODN supplies stability to a lipid nanoparticle complex by addinglipophilic interaction in a system otherwise solely held together byelectrostatic interaction. In addition, this lipophilic additionpromotes cell permeation by localizing the ODN to the outer leaflet ofthe cell membrane. Applying a peptide such as gramicidin or JTS-1further promotes cell permeation of the formulation due to its fusogenicproperties. Alternatively, addition of an enzyme such as proteinase Kcould further aid the ODN in resisting degradation.

Depending on the therapeutic application, the lipid nanoparticlesdescribed herein may be administered by the following methods: peroral,parenteral, intravenous, intramuscular, subcutaneous, intraperitoneal,transdermal, intratumoral, intraarterial, systemic, orconvection-enhanced delivery. In particular embodiments, the lipidnanoparticles are delivered intravenously, intramuscularly,subcutaneously, or intratumorally. Subsequent dosing with different orsimilar lipid nanoparticles may occur using alternative routes ofadministration.

Pharmaceutical compositions of the present disclosure comprise aneffective amount of a lipid nanoparticle formulation disclosed herein,and/or additional agents, dissolved or dispersed in a pharmaceuticallyacceptable carrier. The phrases “pharmaceutical” or “pharmacologicallyacceptable” refers to molecular entities and compositions that produceno adverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human. The preparation of apharmaceutical composition that contains at least one compound oradditional active ingredient will be known to those of skill in the artin light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 2003, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standards.

A composition disclosed herein may comprise different types of carriersdepending on whether it is to be administered in solid, liquid oraerosol form, and whether it need to be sterile for such routes ofadministration as injection. Compositions disclosed herein can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally, inutero, orally, topically, locally, via inhalation (e.g., aerosolinhalation), by injection, by infusion, by continuous infusion, bylocalized perfusion bathing target cells directly, via a catheter, via alavage, in cremes, in lipid compositions (e.g., liposomes), or by othermethod or any combination of the forgoing as would be known to one ofordinary skill in the art (see, for example, Remington's PharmaceuticalSciences, 2003, incorporated herein by reference).

The actual dosage amount of a composition disclosed herein administeredto an animal or human patient can be determined by physical andphysiological factors such as body weight or surface area, severity ofcondition, the type of disease being treated, previous or concurrenttherapeutic interventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a composition herein and/or additional agents isformulated to be administered via an alimentary route Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier.

In further embodiments, a composition described herein may beadministered via a parenteral route. As used herein, the term“parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered, for example but not limited to, intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally (U.S. Pat. Nos. 6,753,514, 6,613,308,5,466,468, 5,543,158; 5,641,515; and 5,399,363 are each specificallyincorporated herein by reference in their entirety).

Solutions of the compositions disclosed herein as free bases orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption such as,for example, aluminum monostearate or gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety, and purity standards as required by FDAOffice of Biologics standards.

Sterile injectable solutions are prepared by incorporating thecompositions in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. Generally, dispersions are prepared byincorporating the various sterilized compositions into a sterile vehiclewhich contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, some methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. A powderedcomposition is combined with a liquid carrier such as, e.g., water or asaline solution, with or without a stabilizing agent.

In other embodiments, the compositions may be formulated foradministration via various miscellaneous routes, for example, topical(i.e., transdermal) administration, mucosal administration (intranasal,vaginal, etc.) and/or via inhalation.

Pharmaceutical compositions for topical administration may include thecompositions formulated for a medicated application such as an ointment,paste, cream or powder. Ointments include all oleaginous, adsorption,emulsion and water-soluble based compositions for topical application,while creams and lotions are those compositions that include an emulsionbase only. Topically administered medications may contain a penetrationenhancer to facilitate adsorption of the active ingredients through theskin. Suitable penetration enhancers include glycerin, alcohols, alkylmethyl sulfoxides, pyrrolidones and luarocapram. Possible bases forcompositions for topical application include polyethylene glycol,lanolin, cold cream and petrolatum as well as any other suitableabsorption, emulsion or water-soluble ointment base. Topicalpreparations may also include emulsifiers, gelling agents, andantimicrobial preservatives as necessary to preserve the composition andprovide for a homogenous mixture. Transdermal administration of thecompositions may also comprise the use of a “patch.” For example, thepatch may supply one or more compositions at a predetermined rate and ina continuous manner over a fixed period of time.

In certain embodiments, the compositions may be delivered by eye drops,intranasal sprays, inhalation, and/or other aerosol delivery vehicles.Methods for delivering compositions directly to the lungs via nasalaerosol sprays has been described in U.S. Pat. Nos. 5,756,353 and5,804,212 (each specifically incorporated herein by reference in theirentirety). Likewise, the delivery of drugs using intranasalmicroparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts and could be employed to deliverthe compositions described herein. Likewise, transmucosal drug deliveryin the form of a polytetrafluoroetheylene support matrix is described inU.S. Pat. No. 5,780,045 (specifically incorporated herein by referencein its entirety), and could be employed to deliver the compositionsdescribed herein.

It is further envisioned the compositions disclosed herein may bedelivered via an aerosol. The term aerosol refers to a colloidal systemof finely divided solid or liquid particles dispersed in a liquefied orpressurized gas propellant. The typical aerosol for inhalation consistsof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

EXAMPLES

Certain embodiments of the present invention are defined in the Examplesherein. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

Materials and Methods

Materials

1,2-Dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA) was obtained fromCorden Pharma (Boulder, Colo., USA).1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased fromAvanti Polar Lipids (Alabaster, Ala., USA). Cholesterol (CHOL) andCremephor EL were obtained from Sigma Aldrich (St. Louis, Mo., USA).N-(carbonyl-methoxypolyethyleneglycol2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG) waspurchased from NOF America Corp. (White Plains, N.Y., USA). The AM-21sequence, u*c*a*acaucagucugauaag*c*u*a (SEQ ID NO: 1), where lower caseletters represent 2′-O-methyl bases and asterisks representphosphorothioate linkages, was obtained from Alpha DNA (Montreal,Quebec, CA) at desalted purity. PrimeTime qPCR assay primer probes andkits for DDAH1, PTEN, RECK, PDCD4, TIMP3 target genes and GAPDHhousekeeping gene were purchased from Integrated DNA Technologies(Coralville, Iowa, USA).

Synthesis of QT

QT were prepared by serial ethanol dilution method. Briefly, all lipids(X/Y/36/20/4 mol/mol, DODMA/DOTAP/DOPC/CHOL/DPPE-PEG) were dissolved inethanol and combined with an equal volume of AM-21 dissolved in citricacid buffer (20 mM, pH 4.5), maintaining a 10:1, lipid:AM weight ratio.DODMA and DOTAP content were varied with the total molar percent oftertiary and quaternary amine maintained at 40 molar percent composition(X+Y=40). This solution was further diluted by equivalent volumes (1:1)of citric acid buffer, NaCl/NaOH buffer (300 mM NaOH, 20 mM NaOH, pH7.4), and PBS (10 mM, pH 7.4). The resultant LN solution wasconcentrated by tangential diafiltration to remove excess ethanol and toreach the appropriate final concentration. Samples were stored at 4° C.prior to characterization.

Mean Particle Diameter and Surface Charge

Aliquots of QT/AM-21 were diluted in PBS. Particle size was measured bydynamic light scattering (DLS) on a NICOMP 370 Submicron Particle Sizer(NICOMP, Santa Barbara, Calif., USA). Aliquots of QT/AM-21 or complexescontaining only tertiary or quaternary amine were diluted in citric acidbuffer or PBS to demonstrate the pH dependency of surface charge. Zetapotential measurement was conducted on a Zeta PALS Analyzer (BrookhavenInstruments Corp., Worcestershire, N.Y., USA).

Drug Loading and Stability

Encapsulation efficiency was determined by Quant-iT™” Ribogreen RNAassay kit (Life Technologies, Carlsbad, Calif., USA). Briefly, QT/AM-21complexes were lysed with Triton X-100 and mean fluorescent intensitywas compared with intact QT/AM-21 at (480 nm λ_(ex), 520 nm λ_(em)).Relative encapsulation efficiency was determined with the formula:(%)=(1FI _(without Triton X-100) /FI _(with Triton X-100))×100%.

Formulation stability was evaluated at −20, 4, and 25° C. over a periodof 30 days. The particle size was periodically monitored by DLS. 10%sucrose was added as a cryoprotectant prior to storage.

Cell culture

A549 cells were purchased from the American Type Culture Collection(Rockville, Md., USA) and grown in RPMI 1640 (Corning, Tewksbury, Mass.,USA) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich, St.Louis, Mo., USA) and 100 units/mL penicillin and 100 mg/mL streptomycin.Cells were maintained at 37° C. and grown under a humidified atmospherecontaining 5% CO₂.

In Vivo Gene Regulation

Cells were grown in 24-well plates at a density of 7.0×10⁵ cells/well 24h prior to transfection. QT/AM-21 of varying lipid composition orQT/negative control (NC) were administered at 50 nM in the presence of20% serum containing media to determine the optimal QT composition.QT/AM-21 was also tested at 1.56, 6.25, 25, and 100 nM to demonstratedose dependency of treatment. Cells were incubated at 37° C. withtransfection media for 4 h and then washed three times with PBS. Freshcomplete cell culture media was added and the cells were incubated at37° C. for an additional 44 h. RNA was isolated from cells by RNeasy 96kit (Qiagen, Valencia, Calif., USA). qRT-PCR was conducted with Taqman®MicroRNA Assay (Life Technologies) or EXPRESS One-Step Superscript®qRT-PCR kit (Life Technologies) on an Applied Biosystems StepOnePlus™RT-PCR system (Life Technologies). The relative amount of DNA wascalculated and compared according to the 2^(−ΔΔCt) method.

Cell Viability Assay

Cells were grown in 96-well plates at a density of 2.0 ×10⁴ cells/well.Cells were treated with controls or QT/AM-21 at 50, 100, or 200 nM, withand without PTX (4.1 nM) dissolved in a small volume of 1:1 CremophorEL:ethanol solution. Relative cell viability was quantified by CellTiter96® AQueous One Solution Cell Proliferation Assay (Promega, Madison,Wis., USA) according to the manufacturer's protocol 72 h following thestart of treatment. Briefly, 20 μL MTS assay solution was added to eachwell and the plates were incubated for 1 h. The absorbance at 490 nm wasrecorded to determine cytotoxicity relative to the untreated control.

Migration Assay

A wound healing model was conducted to examine the migratory ability ofA549 cells following treatment. A549 cells were plated at a density of6.0×10⁵ cells/well in a 33 mm petri dish 24 h prior to transfection. Ascratch wound across the dish was made using a 10 μL pipet tipimmediately before treatment. Culture media was removed and replacedwith transfection media containing QT/AM-21 or appropriate controlsdiluted in complete media. Cells were allowed to proliferate at 37° C.for 48 h. Distances between edges of the wound were measured on a NikonE800 microscope (Nikon, Tokyo, Japan) and SPOT Advanced Imaging Software(v5.0, Diagnostic Instruments Inc., Sterling Heights, Mich., USA).

Invasion Assay

Matrigel (BD Biosciences, San Jose, Calif., USA) was combined withserum-free RPMI 1640 culture media in a 1:1 ratio. 70 μL of gel wasadded to each well insert of a 24-well plate. The gel was allowed to setfor 1 h at 37° C. A549 cells were seeded at 7.5×10⁵ cells/well in avolume of 100 μL/well on top of the gel in the insert. Transfectionmedia containing various formulations or controls at 2×concentration ina 100 μL volume were added to the top of the well inserts. 500 μL 10%fetal bovine supplemented media was added as a chemoattractant below thetranswell insert. The plate was incubated at 37° C. for 48 h. Followingthe incubation period, cells remaining in the top of the well insertswere removed with a cotton swab. Well inserts were rinsed with PBS andplaced in 500 uL 0.25% trypsin solution for 1 h at 37° C. Detached cellswere counted on a hemocytometer.

Tumor Regression Analysis

A549 mouse xenograft models were generated by inoculating female athymicnude mice with 1.0×10⁶ cells/mouse. Tumors were allowed to reach a sizeof ≥100 mm³ before treatment began (˜2 weeks). Mice (n=10 per group)were dosed by tail vein injection with saline control, 0.5, or 1 mg/kgQT/AM-21. Tumor progression was routinely monitored through the courseof the study. Tumor volume was calculated according to the formula:V=(L·W²)/2. Mice were dosed every three days for the first threetreatments and then once a week following the first dose for a total ofseven doses. All mice were treated according to the guidelines deemedappropriate by the Institutional Animal Care and Use Committee (IACUC)of the Ohio State University (OSU).

Combination Therapy Analysis

Female athymic nude mice (n=5 per group) were implanted with 1.0×10⁶A549 cells/mouse and treatment began when tumors reached a size of ≥100mm³. Mice were treated by tail vein injection with saline control or 1mg/kg QT/AM-21. Mice receiving PTX treatment as monotherapy orcombination therapy received PTX dissolved in 1:1 Cremephor EL:ethanolsolution at a dose of 3 mg/kg via intraperitoneal injection. Mice weredosed on days 1, 3, 5, 8, 15, 22, 29 and were monitored over a 4 weekperiod. 48 h following the last dose, mice were euthanized and tumorswere collected for gene regulation study.

In Vivo Gene Regulation

Tumors were harvested and placed in TRIzol reagent (Life Technologies)following treatment and homogenized. mRNA was isolated per themanufacturer's protocol. qRT-PCR was then completed according to thesame procedure as outlined in the in vitro section.

Statistical Analysis

All studies were done in triplicate unless otherwise mentioned.Statistical analysis was conducted on Microscoft Excel Software (2013,Redmond, Wash., USA). Student's t-test was used to determine statisticalsignificance between two or more groups. p≤0.05 was selected as thecutoff for statistical significance.

Example 1

Increased Expression of Target Genes in “QTsome™” Lipid NanoparticleFormulation Formulated Anti-miR-21 (AM-21) Treated KB Cell In Vitro

KB cells (Hela derivative cancer cells) were cultured in vitro andtransfected with free, unformulated AM-21, or “QTsome™” lipidnanoparticle formulation that is formulated with AM-21. Two anti-miR-21oligonucleotides were used for “QTsome™” lipid nanoparticle formulationand transfection (AM-21a and AM-21b).

AM-21a: (SEQ ID NO: 1) 5′-u*c*a*acaucagucugauaag*c*u*a-3′; and AM-21b:(SEQ ID NO: 2) 5′-5′-u*c*a*acaucagucugauaag*c*u*a-CHOL-3′.

In either AM, all bases are 2′O-methylated. *represent phosphorothioatelinkages.

The transfection efficiency was measured and compared. As shown in FIG.1, “QTsome™” lipid nanoparticle formulation greatly enhance thetransfection of AM-21 oligonucleotide into KB cells, where in FIG. 1,the Y-Axis is PTEN expression level (%).

Example 2

Increased Expression of Target Genes in “QTsome™” Lipid NanoparticleFormulation Formulated Anti-miR-21 (AM-21) Treated Lung Cancer Cells InVitro

Lung cancer A549 cells (human lung adenocarcinoma epithelial cell line)were cultured in vitro as standard cell culture. Cells were cultured inRPMI 1640 media supplemented with 10% FBS and 1%antibiotics/antimycotics in a 5% CO₂ atmosphere at 37° C.

The lung cancer A549 cells were then treated with either free,unformulated anti-miR-21 (AM-21, 5′-U*C*A*ACAUCAGUCUGAUAAG*C*U*A-3′, SEQID NO:3), or “QTsome™” lipid nanoparticle formulation formulatedanti-miR-21 (SEQ ID NO:2), QT/AM-21. The untreated A549 cells were usedas control.

A549 cells were grown to 70% confluency. Serum-free transfection mediacontaining 100 nM AM-21 was added to cells for 4h. Followingtransfection, fresh serum-containing culture media was added to cells.AM-21b: 5′-5′-u*c*a*acaucagucugauaag*c*u*a-CHOL-3′ (SEQ ID NO. 2) wasused.

To confirm the effect of anti-miR-21 treatment on inhibiting miR-21activity in A549 cells, the expression levels of two target genes ofmiR-21: RECK and PTEN were measured after AM-21 treatment.

RNA was extracted by TRIzol reagent per manufacturer's protocol. cDNAwas generated by Maxima First Strand cDNA synthesis kit permanufacture's protocol. RT-PCR was completed on an Applied BiosystemsStepOne Plus system using Luminaris Color HighGreen High ROX qPCR MasterMix according to the manufacturer's protocol. RECK and PTEN mRNA levelswere measured relative to GAPDH.

As shown in FIG. 2A and FIG. 2B, A549 cells (NSCLC) were transfected 4 hwith 100 nM AM-21. The level of miR-21 decreased following treatment byover 40%. QT/AM-21 demonstrate over 2.5-fold upregulation for PTEN andDDAH1 and moderate upregulation for RECK, PDCD4, and TIMP3. That is, theexpression of RECK in both free AM-21 and QT/AM-21 treated A549 cells isupregulated, and the expression level in QT/AM-21 treated A549 cells isabout 2.75 times higher, as compared to the expression in untreatedcontrol A549 cells. It is also shown that the expression of PTEN isupregulated about 1.5 times only in QT/AM-21 treated A549 cells. Thedata indicate anti-miR-21 is effective on inhibiting the activity ofanti-miR-21, resulting in increased expression of its target mRNAs. FIG.2B shows that the upregulation of target genes occurs in a dosedependent manner for DDAH1, PTEN, and RECK.

Thus, treatment with 100 nM QT/AM-21 resulted in moderate to strong downregulation of miR-21 and upregulation of its gene targets (FIG. 3A, FIG.2B). Relative to the untreated control, miR-21 in the treated groupdecreased by 50.3±2.1% following administration of QT/AM-21. Little tono effect on target gene regulation was observed for the scrambled NCgroup. Tumor suppressors PTEN and PDCD4 were upregulated 2.7 and1.3-fold respectively. Matrix metalloprotease inhibitors RECK and TIMP3were both upregulated by approximately 1.5-fold. Migration inhibitorsANKRD46 and DDAH1 were upregulated 1.2 and 3.0-fold respectively.

FIG. 2C shows dose dependency results where varying dosages of QT/AM-21between 1.56 to 100 nM were administered and qRT-PCR was conducted toevaluate the relationship between AM concentration and miR-21 targetgene upregulation. DDAH1, PTEN, and RECK all demonstrated a directcorrelation between dose and response. DDAH1 and PTEN were relativelymore sensitive to changes in concentration relative to RECK.

Example 3

Synergistic Effects of QT/AM-21 with other Anti-cancer Treatments

A549 cells were cultured as in Example 2, and treated with eitherQT/AM-21 (5′-U*C*A*ACAUCAGUCUGAUAAG*C*U*A-3′, SEQ ID NO:3) at threedifferent concentrations (50 nM, 100 nM or 200 nM), paclitaxel (PTX) ora combination of PTX and QT/AM-21 (PTX/50 nM QT/AM-21; PTX/QT/100 nM-21;TX/200 nM QT/AM-21). (Dosage of PXT was 4nM).

The cells were incubated with the drugs for 72 h; after which the cellswere harvested and tested for cytotoxicity and viability using MTS assaykit by Promega. Absorbance scanning was done at 490 nm to determinerelative cell viabilities.

The data show that free, unformulated AM-21 and “QTsome™” lipidnanoparticle formulation do not confer significant cytotoxicity. Onlythe higher concentration (200 nM QT/AM-21) treatment generates somedegree of cytotoxicity.

As shown in FIG. 3A and FIG. 3B, about 70% and 65% of cancer cellstreated with 200 nM QT/AM-21 and PTX respectively, as compared to theuntreated control cancer cells, are still alive, while the effect of thecombined treatment with QT/AM-21 and PTX is statistically significantlystronger than either individual treatment (p<0.05), leading only about35% of cells alive after the treatment. These data show that QT/AM-21 iscapable of increasing the chemosensitization in cancer cells, e.g., lungcancer cells.

FIG. 3C is a set of photographs showing sensitivity data with andwithout paclitaxel (PTX) for untreated, anti-miR21 (AM-21) (200 nM);“QTsome™” lipid nanoparticle formulation (lipids), “QTsome™” lipidnanoparticle formulation/AM-21 (50 nM); “QTsome™” lipid nanoparticleformulation/AM-21 (100 nm); and “QTsome™” lipid nanoparticleformulation/AM-21 (200 nM). The combination of PTX and QTsome™/AM-21results in substantial gains of cytotoxicity, as indicated by thechanges in cellular morphology.

Thus, FIGS. 3A-3C show cell viability where treatment with free AM-21 orQT lipids did not result in significant cytotoxicity as analyzed by MTSassay (FIG. 3A). Likewise, the combination of QT/AM-21 at 50 nM did notdemonstrate much cytotoxicity. However, moderate increases incytotoxicity were observed at increased concentrations of QT/AM-21 (100,200 nM). Addition of PTX alone diminished cell viability by 40%.Addition of free AM-21 or QT lipids to PTX did not result in significantdecreases in cell viability. However, substantial gains in cytotoxicitywere attributed to the addition of QT/AM-21 at increasing doses. Cellviability was reduced to 51.2%, 41.0%, and 31.8% for 50, 100, and 200 nMdoses, respectively. The difference between untreated and QT/AM-21groups was not significant, but differences between the QT/AM-21 and PTXmonotherapies and the combination therapy had p<<0.05. Changes in cellmorphology were also noted by microscopy, as seen in FIG. 3B. Minorchanges in cellular morphology were observed for control treatmentgroups and for low doses of QT/AM-21. Major alterations in morphologywere observed corresponding to increasing cytotoxicity for increasingdoses of QT/AM-21 and especially QT/AM-21 with PTX.

Example 4

QT/AM-21 Inhibits miR-21 in the KB Cell Subcutaneous Xenograft

Mice (Athymic Ncr-nu/nu) were inoculated subcutaneously with KB cell toestablish KB subcutaneous xenograft tumor models. Mice were inoculatedwith 1×10⁶ cells/mouse. Mice were allowed to develop over a two weekperiod prior to treatment. Mice with developed tumor at certain size(about 50mm³) were administered with four doses of 2 mg/kg QT/AM-21(N=2) (days 1, 3, 7 and 10) by intravenous (i.v.) injection. Aftertreatment, tumor tissues were dissected and the expression levels ofPTEM, RECK and TIMP3 were measured. The expression levels of all thethree genes in QT/AM-21 treated mice are increased, as compared to theirexpression levels in the untreated mice (N=7), as shown in FIG. 4A.

During the course of treatment, tumor growth was measured and comparedbetween the QT/AM-21treated mice and control mice. The data in FIG. 4Bshow significant inhibition of tumor growth in QT/AM-21 treated mice(p<0.05).

Example 5

QT/AM-21 Induces Tumor Regression in A549 Subcutaneous Xenograft

Mice (Athymic Ncr-nu/nu) were inoculated subcutaneously with A549 cellsto establish subcutaneous xenograft tumor models. Mice were inoculatedwith 1×10⁶ cells/mouse. Mice were randomized into two groups of sevenwhen tumor volume reached more than 50 mm³ (about two weeks afterimplantation of A549 cancer cells). QT/AM-21 was administered to mice(N=7) at 2 mg/kg by i.v. injection three times in the first week (days1, 4 and 7) and once a week thereafter (days 14 and 21). Tumor volumesfrom treated and untreated (N=7) mice were then measured every otherdays, as shown in FIG. 5A. Significant decrease in tumor size isachieved through treatment with QTsome™/AM-21 (1 mg/kg)

The data show that QT/AM-21 treatment induces tumor regression in A549subcutaneous xenograft model. Furthermore, tumors from QT/AM-21 treatedmice (N=3) and control mice (N=7) were weighted at the end of treatment(day 24). The tumor weight from QT/AM-21 treated mice is significantlylighter than that from control mice (p<0.00001), as shown in FIG. 5E.Except for the differences in tumor weight, no significant differenceswere observed between untreated control mice and QT/AM-21 treated micein terms of liver (p=0.5360) (FIG. 5C), and spleen (p=0.9832) weight(FIG. 5D). Only moderate degree of difference in body weight is observedbetween two groups (p=0.0468), as shown in FIG. 5B.

FIG. 5F shows the Kaplan-Meier survival, as for untreated, and QT/AM-21(0.5 mg/kg, 1.0 mg/kg). Treatment with 1 mg/kg QT/AM-21 substantiallyincreases survival time of A549 xenograft mice.

In a combination therapy protocol, mice were given 1 mg/kg QT/AM-21and/or 3 mg/kg PTX. As seen in FIG. 5G, treatment with a combination ofPTX and QT/AM-21 is able to achieve greater tumor suppression thaneither agent administered as monotherapy.

In vivo gene regulation of DDAH1 and PTEN are shown in FIG.5H and FIG.51, respectively. Treatment with PTX achieves moderate upregulation ofDDAH1 and PTEN, while QT/AM-21 brings about strong upregulation. Thecombination of PTX and QT/AM-21 result in greater gene upregulation thantreatment with either agent alone.

In sum, the QTsome™ lipid nanoparticle formulations exhibit smallparticle size, moderate zeta potential, high drug loading, and long termstability. In vitro analyses indicate strong, dose-dependentupregulation of miR-21 targets as well as increased sensitivity topaclitaxel and reduced migration and invasion with QT/AM-21 treatment.In vivo analyses reveal tumor regression, upregulation of target genes,enhanced anticancer activity with combination therapy, and prolongedsurvival.

Thus, treatment with QT/AM-21 demonstrates strong anti-tumor activity(FIG. 5A) at 1 mg/kg, but diminished activity at 0.5 mg/kg. Treatment oftumors initiated at ˜180 mm³ and ended at 816.75, 618.125, and 172.5 mm³for the untreated, 0.5 mg/kg, and 1 mg/kg groups respectively. Moderatedifferences in terms of body weight were observed between the treatedand untreated groups, with a difference of about 1.5 g. Liver and spleenweights remained fairly consistent between the two groups, suggestinglittle to no toxicity for these organs. Tumor weight was over 16-foldlower for the treated group compared to the untreated group (FIGS.5B-5E). In terms of median survival time, the untreated group was 21days, the 0.5 mg/kg treated group was 24 days, and the 1 mg/kg treatedgroup was significantly prolonged, at 33 days (FIG. 5F).

With respect to in vivo combination therapy, PTX and QT/AM-21combination therapy was evaluated for therapeutic efficacy. Treatmentbegan when tumors reached ˜80 mm³ in volume. Tumors progressed to 380,246.4, 201.1, and 138.1 mm³ for the untreated, PTX, QT/AM-21, andcombination treatment groups respectively (FIG. 5G). Furthermore, qPCRconducted on tumor sections revealed moderate to strong upregulation ofDDAH1 and PTEN (FIG. 5I). DDAH1 and PTEN were only modulated slightly byPTX, 1.7 and 1.5-fold respectively. DDAH1 was upregulated 3.4-fold whilePTEN was upregulated 2.5-fold with QT/AM-21. DDAH1 was stronglyupregulated by 5-fold and PTEN was upregulated 4.1-fold with thecombination therapy.

Example 6

QT/AM-21 Reduces Migration and Invasion of A549 Cells In Vitro

A549 cells were cultured following standard culture procedure andincubated with QT/AM-21 (5′-U*C*A*ACAUCAGUCUGAUAAG*C*U*A-3′, SEQ IDNO:3) at three different concentrations (50 nM, 100 nM and 200 nM), orfree, unformulated AM-21 (200 nM) for 48 hours. The untreated cells andcells treated with empty “QTsome™” lipid nanoparticle formulation wereused as control. The migration of tumor cells was determined. The resultdemonstrates that QT/AM-21treatment reduces tumor cell migration, asshown in FIG. 6A.

The invaded tumor cells in QT/AM-21 treated culture are significantlower, as compared to the untreated cells or cells only treated withempty “QTsome™” lipid nanoparticle formulation lipid nanoparticles(about 60 Vs about 100) (Number of cells passing through the gelcoating), as shown in FIG. 6B.

The data also show that QT/AM-21 is dose dependent as higher treatmentlevel generates a more significant effect on both cell migration andinvasion (FIG. 6A and FIG. 6B). (Note: the AM-21 used in Examples was:5′-U*C*A*ACAUCAGUCUGAUAAG*C*U*A-3′, (SEQ ID NO:3) where the sequencecontains phosphorothioate linkages (*)).

Thus, the in vitro scratch wound healing assay shows the relativemobility of A549 following treatment with AM-21. QT lipids or AM-21alone did not confer significant decreases in cell migration in thewound region. With 100 and 200 nM treatment, mobility was reduced to43.0 and 22.2% relative to the untreated control (FIG. 6A). Matrigel isoften used to simulate biological conditions of the basement membrane.The ability of cancer cells to migrate plays a role in determiningmetastatic potential and increases with cancer progression. Treatmentwith QT/AM-21 at 50, 100, and 200 nM were able to reduce migration to87.7, 76.7, and 62.6% respectively, while QT lipids or AM-21 did notsignificantly retard cell invasion (FIG. 6B).

Example 7

QTSome Formulations

“QTsome™” lipid nanoparticle formulation can significantly increase theeffect of AM-21 on the reduction of tumor cell migration, as compared tothe free, unformulated AM-21.

A matrigel invasion assay was also performed after 48 hour treatment, asfollows: Chilled Matrigel was combined with chilled serum-free RPMI 1640culture media in a 1:1 ratio. 70 uL of gel was added to each well insertof a 24-well plate. The gel was allowed to set for 1 hour at 37° C. A549cells were seeded at 75,000 cells/well in a volume of 100 uL/well on topof the gel. Transfection media containing various formulations orcontrols at 2×concentration in a 100 uL volume were added to the wellinserts. The plate was incubated at 37° C. for 48 hours. Following theincubation period, cells remaining in the top of the well inserts wereremoved with a cotton swab. Well inserts were rinsed with PBS and placedin 500 uL 0.25% trypsin solution for 1 hour at 37° C. Detached cellswere counted on a hemocytometer.

FIG. 7 depicts the “QTsome™” lipid nanoparticle formulation mechanism ofaction. Under pH 7.4, quaternary amine-cationic lipids (QA-CLs) maintain“+” charge to provide stability. Under pH 5.5, both QA-CLs and tertiaryamine-cationic lipids (TA-CLs) are charged, which promotes endosomemembrane interaction/disruption.

FIG. 8 depicts the relative luciferase expression of combinations oftertiary and quaternary cationic lipids. At a Q-to-T amine-cationiclipid ratio of 5:40, over 85% downregulation is demonstrated forluciferase siRNA transfection in HCC cells expressing luciferase. Forthe development of “QTsome™” lipid nanoparticle formulation, lipid stocksolutions were created by dissolving lipids in 100% ethanol. All lipidswere obtained from Avanti Polar Lipids (USA) or Sigma Aldrich (USA) andused without further purification. Lipids (egg phosphatidylcholine:cholesterol: TPGS, 15:35:5) were combined with varying concentrations oftertiary (DODMA) and quaternary (DOTMA) amine (45:0, 5:40, 15:30,22.5:22.5, 30:15, 40:5, 45:0; DODMA:DOTMA) in 1.0 mL vials. Additionalethanol was added to reach a volume of 180 μL. This was then combinedwith 420 μL 10 mM citric acid buffer to reach a final concentration of30% ethanol. The formulations were combined with SILENCER FireflyLuciferase (GL2+GL3) siRNA (Invitrogen) at an amine to phosphate (N:P)ratio of 15:1. Formulations were allowed to incubate for 15 minutesprior to dilution with serum-free DMEM (GIBCO) to a total volume of 300μL. Lipofectamine 2000 (Invitrogen) was used as a positive control andcombined with the same amount of siRNA at the same N:P and diluted tothe same total volume.

SK-HEP-1 (hepatocellular carcinoma) cells expressing luciferase, grownin DMEM medium at 37° C. under 5% CO₂ atmosphere, were plated 24 h priorto transfection at a density of 2×10⁴ cells per well in a 96-well plate.Cells were grown to approximately 80% confluency and the serumcontaining media was removed. Cells were transfected with 70 μLtransfection media and treated for 4 h. Experiments were performed withfour replicates. After treatment was completed, cells were washed with1X PBS and serum-containing DMEM was restored. 48 h after treatment wascompleted, cells were analyzed for luciferase expression by a LuciferaseAssay Kit (Promega) per the manufacturer's instructions. The results areshown in FIG. 8. The most efficacious transfection activity wasexhibited by the formulation containing 5% DOTMA and 40% DODMA, showingover 85% knockdown in luciferase expression.

FIG. 9 is a schematic illustration showing a design of “QTsome™” lipidnanoparticle formulation with a list of lipid nanoparticle components:tertiary amine: 1,2-dioeyloxy-N,N-dimethyl-3-aminopropane (DODMA);quaternary amine: 1,2-dioleoyl-3-dimethylammonium-propane (DOTAP);neutral lipid: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); helperlipid: cholesterol (CHOL); and PEGylating agent:N-(carbonyl-methoxypolyethyleneglycol2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG).

FIG. 10A is a schematic illustration of an example of synthesis of“QTsome™” lipid nanoparticle formulation, as follows:

In a first step, lipids in 100% EtOH and anti-miR (AM) in citric acidbuffer are combined to form “QTsome™” lipid nanoparticle formulation in50% EtOH.”

In a second step, the “QTsome™” lipid nanoparticle formulation in 50%EtOH” is diluted at a 1:1 ratio with 20 nM citric acid buffer, pH 4.5 toform “QTsome™” lipid nanoparticle formulation in 25% EtOH.”

In a third step, the “QTsome™” lipid nanoparticle formulation in 25%EtOH” is diluted at a 1:1 ratio with 300 nM NaC1, 20 nM NaOH buffer, pH7, to form “QTsome™” lipid nanoparticle formulation in 12.5% EtOH.”

In a fourth step, the “QTsome™” lipid nanoparticle formulation in 12.5%EtOH” is diluted at a 1:1 ratio with PBS, pH 7, to form “QTsome™” lipidnanoparticle formulation in 6.25% EtOH.”

In a fifth step, the “QTsome™” lipid nanoparticle formulation in 6.25%EtOH” is subjected to diafiltration to concentrate “QTsome™” lipidnanoparticle formulation and remove EtOH, to form “QTsome™” lipidnanoparticle formulation in ˜0% EtOH.”

FIG. 10B shows where the ratios of a quaternary: tertiary(% mol) wereadjusted to optimize one formulation. Based upon physicalcharacterization and prior in vitro transfection data, 15:25 wasselected for the base composition of the QTsome™” lipid nanoparticleformulation.

In certain embodiments, it is desired to have small particles (<150 nm)and moderate zeta potential (+10-30 mV) to potentiate delivery ofoligonucleotides. Furthermore, it is desirable that the lipidnanoparticles exhibit high stability and slow release to maximize invivo delivery and therapeutic efficacy. Particle size measurement by DLSindicated particles of approximately 80-170 nm in diameter (FIG. 10B).Particles with greater amount of quaternary amine (15-40 mol %) achievedparticles of smaller size (<120 nm).

FIG. 11 is a graph showing the effect of pH on surface charge, comparingparticle size and zeta potential. In certain embodiments, the particlesize is less than about 150 nm to facilitate EPR effect and to allow forpost-production sterile filtration. Also, in certain embodiments, thezeta potential is moderate to avoid nonspecific uptake by macrophagesand (reticuloendothelial system (RES) mediated clearance. In the datashown in the graph in FIG. 11, the mean particle diameter is104.9+/−54.8 nm. With respect to the zeta potential, placing “QTsome™”lipid nanoparticle formulation under acidic pH cationizes the tertiaryamine, leading to increase surface charge.

Zeta potential measurement (FIG. 11) revealed QT particles of 12.49±1.45mV in PBS (pH 7.4) and 29.89±8.16 mV in citric acid buffer (pH 4.0),thus demonstrating the pH responsive behavior of the conditionallyionizable formulation and potential application in promoting endosomallysis. The pH responsive behavior of QT fell between the range ofcharges for lipid nanoparticles containing only tertiary or quaternaryamine at similar pH values. The charge on tertiary amines changed from 3to 15 mV with increasingly acidic pH. Conversely, quaternary aminecontaining lipid nanoparticles did not vary much with changes in buffersolution (27 and 29 mV). This intermediate pH-responsive property maythus be desirable in certain embodiments for desired in vivo activity ofQT.

FIG. 12A is a graph showing drug loading efficiency. The encapsulationefficiency(%)=(1−FI_(without Triton X-100)/FI_(with Triton X-100))×100%. Theencapsulation efficiency was determined to be 83.3+/−4.17% by OligreenAssay. The CL4B column separation of “QTsome™” lipid nanoparticleformulation further provides evidence of nearly complete anti-miR (AM)encapsulation.

Encapsulation efficiency studies demonstrated high drug loading of83.3±4.17% by fluorescent intensity measurement. CL 4B gel-filtrationchromatography analysis (FIG. 12A) showed approximately 90%encapsulation of the oligonucleotide within the encapsulated drugfractions with very little oligonucleotide remaining in the free drugfractions.

FIG. 12B is a graph showing particle storage stability, comparing meandiameter (nm) at: −20° C. (top line); 4° C. (bottom line); and, 25° C.(middle line) over time (days).

The formulation further demonstrate high stability under storage at −20(with 10% sucrose as a cryoprotectant), 4, and 25° C. over a period of30 days, maintaining a size of −110 nm (FIG. 12B).

FIG. 13 is a graph showing one example of formula optimization where aseries of QT formulations were evaluated to determine the differentdesirable ratios of quaternary and tertiary lipoamine for transfection.Formulations are identified as QT(mol % quaternary amine)-(mol %tertiary amine) in FIG. 13. Treatment with 50 nM AM-21 revealed QT with15 mol % DOTAP and 25 mol % DODMA to perform best in upregulation ofDDAH1 (1.33-fold). This combination of lipids was chosen for further invitro and in vivo experiments, as described herein. Formulations QT10-30and QT5-35 also performed well, with 1.32 and 1.27-fold upregulationrespectively. Formulations containing only quaternary or tertiarylipoamine did not perform as well relative to the formulations with thevarious combinations.

FIG. 14 is a graph showing stability, as measured by an in vitro releaseprofile, comparing percent drug released over time (hours). The particletend to aggregate over time when stored at −20° C., but are stable whenstored at 4° C. and 25° C. In addition, dialysis against PBSdemonstrates a relatively slow drug release kinetics.

Example 8

FIG. 15 provides a list of anti-miR-21 compounds 101-125 (SEQ IDNOS:4-28) which were designed and tested:

Example 8a

FIG. 16A is a graph showing activity of compounds 116, 118, 117, 112,113, 114, 108, 109, 110, a negative control, and AM-21 (anti-21), inANKRD46 (ANKRD46/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56nM).

FIG. 16B is a graph showing activity of compounds 116, 118, 117, 112,113, 114, 108, 109, 110, a negative control, and AM-21 (anti-21), inDDAH1 (DDAH1/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 16C is a graph showing activity of compounds 116, 118, 117, 112,113, 114, 108, 109, 110, a negative control, and AM-21 (anti-21), inPDCD4 (PDCD4/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8b

FIG. 17A is a graph showing activity of compounds 116, 118, 117, 111,115, 119, 107, 106, 105, a negative control, and anti-AM21, in DDAH1(DDAH1/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 17B is a graph showing activity of compounds 116, 118, 117, 111,115, 119, 107, 106, 105, a negative control, and anti-AM21, in PDCD4(PDCD4/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8c

FIG. 18A is a graph showing activity of compounds 104, 103, 102, 101,120, 121, 122, 123, 124, 125, a negative control, and AM-21 (anti-21),in ANKRD46 (ANKRD46/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56nM).

FIG. 18B is a graph showing activity of compounds 104, 103, 102, 101,120, 121, 122, 123, 124, 125, a negative control, and AM-21 (anti-21),in DDAH1 (DDAN1/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 18C is a graph showing activity of compounds 104, 103, 102, 101,120, 121, 122, 123, 124, 125, a negative control, and AM-21 (anti-21),in PDCD4 (PDCD4/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8d

FIG. 19A is a graph showing activity of compounds 114, 117, 110, 116,118, 109, 121, 101, a negative control, and AM-21, in ANKRD46(ANKRD46/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 19B is a graph showing activity of compounds 114, 117, 110, 116,118, 109, 121, 101, a negative control, and AM-21, in DDAH1 (DDAH1/GAPDH(fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 19C is a graph showing activity of compounds 114, 117, 110, 116,118, 109, 121, 101, a negative control, and AM-21, in PDCD4 (PDCD4/GAPDH(fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8e

FIG. 20A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in ANKRD46(ANKRD46/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 20B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in ANKRD46(ANKRD46/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8f

FIG. 21A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in DDAH1(DDAH1/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 21B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in DDAH1(DDAHUGUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8g

FIG. 22A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PDCD4(PDCD4/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 22B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PDCD4(PDCD4/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8h

FIG. 23A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PTEN(PTEN/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 23B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in PTEN(PTEN/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8i

FIG. 24A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in TIMP3(TIMP3/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 24B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in TIMP3(TIMP3/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

Example 8i

FIG. 25A is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in RECK(RECK/GAPDH (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

FIG. 25B is a graph showing activity of compounds 104, 109, 110, 114,115, 116, 117, 118, 122, AM-21 and a negative control, in RECK(RECK/GUSB (fold change) at 100 nM, 25 nM, 6.25 nM, 1.56 nM).

These examples show how a pH-sensitive carrier, QT is especially usefulfor the delivery of therapeutic oligonucleotides. Tertiary lipoaminesform the pH-sensitive component of QT and upon exposure to acidicconditions as in the endosome, the tertiary lipoamine becomescationized, enabling interaction with negatively charged lipids formingthe endosomal bilayer and consequently leading to efficient endosomalescape of the oligonucleotides. Release of drug from the endosomalcompartment is a critical step in determining drug efficacy. Theinclusion quaternary amine is used to electrostatically stabilize thecharge of the lipid nanoparticles under physiological pH conditions. Thepresence of a significant amount of quaternary amine maintainsinteractions with the negative charge of the oligonucleotides, therebyresulting in particles of smaller average particle size relative toparticles containing mostly tertiary amine content.

In terms of surface charge, one embodiment (QT15-25) displayed anintermediary response to changing buffer condition relative to otherembodiments (QT40-0 or QT0-40). The lipid nanoparticles furthermoredemonstrated excellent colloidal stability and efficient drug loading,thus demonstrating practicality for clinical use and commercialization.Also, in certain embodiments, the QT may be further stabilized by theaddition of cryoprotectant and processing by lyophilization to retainthe drug form's integrity over long term storage.

QT/AM-21 was able to strongly downregulate miR-21 and upregulate severalkey targets of miR-21 including tumor suppressors, matrixmetalloprotease inhibitors, and migration inhibitors. PTX kills cancercells by stabilization of microtubules, which prevents mitosis. It is tobe noted that patients normally respond to PTX with a 40-80% responserate, but many of these patients develop resistance to PTX over time.The combination of QT/AM-21 and PTX demonstrated herein shows greaterreductions in cell proliferation over use of the combination of freeAM-21 and PTX, thus showing greater uptake and/or efficacy of AM-21 whendelivered via QT.

As another example, in an ovarian cancer model, the resistance againstPTX is believed to be regulated by miR-21′s effect on hypoxia-induciblefactor-1α (HIF-1α) and P-glycoprotein (P-gp). HIF-1α and HIF-1β aresubunits of the heterodimeric transcription factor HIF-1. HIF-1α isupregulated in response to oncogene activity, hypoxia, and growthfactors. HIF-1β is comparatively benign and constitutively expressed inthe body. P-gp is a member of the ATP-binding cassette (ABC) transporterfamily, which has been found to play a role in the development ofmultidrug resistance in cancer. As demonstrated herein, decreasedmigratory and invasion potential was observed in a dose dependent mannerin response to QT/AM-21 treatment. Also, it is to be noted that, asanother example, lung cancer is often diagnosed in late in itsdevelopment, when metastasis has already begun. Therefore, the QT/AM-21lipid nanoparticles described herein address a critical need that iscurrently unmet by chemotherapy administered in the late stage.

Further, treatment with QT/AM-21 in a xenograft mouse model was able tosignificantly suppress tumor growth at 1 mg/kg. Targets of miR-21 werealso found to be upregulated, supporting trends observed in vitro. It isto be noted that relative increase in body weight indicated mice withbetter health condition. As demonstrated herein, liver and spleen weightdid not differ much between the two groups, showing that the formulationwas not toxic to those organs. This is an important advantage sinceliver and spleen are heavily fenestrated organs involved in thereticuloendothelial system with large local populations of macrophages,and lipid nanoparticles may accumulate in these organs as a result. Thecombination of PTX and QT/AM-21 demonstrated greater therapeuticactivity than either agent administered alone. Interestingly, while PTXdisplayed greater therapeutic activity in vitro than QT/AM-21, the trendin tumor suppression was opposite and QT/AM-21 was slightly moreeffective than PTX. While not wishing to be bound by theory, it is nowbelieved that the relative increase in QT/AM-21 activity is attributedto increased retention of the lipid nanoparticles in the tumorvasculature following injection, leading to a greater duration oftherapeutic effect.

The lipid nanoparticle formulations described herein provide a broadspectrum of useful application for drug loading and treating otherdiseases. As a large majority of oligonucleotides are roughly 20-25nucleotides in length and encapsulation is sequence independent,dependent instead on the relative anionic charge of the sequence, it ito be understood that the QTsome ™ lipid nanoparticle formulationsdescribed herein are not limited to administration for AM-21. Rather,the QTsome ™ lipid nanoparticle formulations are also useful to delivervirtually any type of AM, siRNA, or miR mimic, with little optimizationrequired.

It is also within the contemplated scope of the present disclosure thatthe addition of a targeting agent may also improve the specificity ofdelivery of QTsome ™ lipid nanoparticle formulations to cancer cell,such as NSCLC cells. One non-limiting example of such additional agentcan be the targeting of epidermal growth factor receptor with cetuximab.

Example 9

FIG. 26A is a table showing the zeta potential without anoligonucleotide for: tertiary amine, quaternary amine, and a QTsome™lipid nanoparticle formulation.

FIG. 26B is a graph showing the zeta potential without anoligonucleotide for: tertiary amine, quaternary amine, and a QTsome™lipid nanoparticle formulation.

Example 10

FIG. 27A is a table showing tumor volume (mm³) over a course of days forsaline and AT/AM-21 (2 mg/kg).

FIG. 27B is a graph showing tumor volume (mm³) over a course of days forsaline and AT/AM-21 (2 mg/kg).

Example 11

FIG. 28A is a table showing tumor volume (mm³) over a course of days forsaline and QT/AM-21 (2 mg/kg), PTX, and a combination of QT/AM-21+PTX.

FIG. 28B is a graph showing tumor volume (mm³) over a course of days forsaline and QT/AM-21 (2 mg/kg), PTX, and a combination of QT/AM-21+PTX.

Example 12

In certain embodiments, the methods provided herein comprise at leastone additional therapy. The at least one additional therapy may comprisea chemotherapeutic agent and/or radiation therapy. The chemotherapeuticagent may be selected from 5-fluorouracil, gemcitabine, doxorubicine,mitomycin c, sorafenib, etoposide, carboplatin, epirubicin, irinotecanand oxaliplatin. The at least one additional therapy may be administeredat the same time, less frequently, or more frequently thanadministration of a composition provided herein.

In any of the methods provided herein, the composition may beadministered once per day, once per week, once per two weeks, once perthree weeks, or once per four weeks.

In any of the methods provided herein, administering results inreduction of tumor size, and/or reduction of tumor number. In any of themethods provided herein, the administering prevents an increase in tumorsize and/or an increase in tumor number. The administering may prevent,stop or slow metastatic progression. The administering may extend theoverall survival time of the subject. The administering may extendprogression-free survival of the subject.

Certain embodiments of the formulations and methods disclosed herein aredefined in the above examples. It should be understood that theseexamples, while indicating particular embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseexamples, one skilled in the art can ascertain the essentialcharacteristics of this disclosure, and without departing from thespirit and scope thereof, can make various changes and modifications toadapt the compositions and methods described herein to various usagesand conditions. Various changes may be made and equivalents may besubstituted for elements thereof without departing from the essentialscope of the disclosure. In addition, many modifications may be made toadapt a particular situation or material to the teachings of thedisclosure without departing from the essential scope thereof.

What is claimed is:
 1. A method of inhibiting miRNA activity in vitro ina cell from a subject or in vivo to a cell in a subject, comprising:introducing an inhibitor composition to a location in vitro or in vivowhere miRNA activity exists, wherein the composition comprises at leastone oligonucleotide encapsulated in a lipid nanoparticle; the lipidnanoparticle being comprised of a combination of permanently ionizedquaternary amine-cationic lipids and conditionally ionized tertiaryamine-cationic lipids; the lipid nanoparticle having a formulationcomprising at least: quaternary amine1,2-dioleoyl-3-dimethylammonium-propane (DOTAP); tertiary amine1,2-dioeyloxy-N,N-dimethyl-3-aminopropane (DODMA); neutral lipid1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); helper lipidcholesterol (CHOL); and, PEGylating agentN-(carbonyl-methoxypolyethyleneglycol2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG),wherein DOTAP and DODMA each constitute 40 mol percent of the totalamount of lipids in the composition, or wherein the lipids in thecomposition consist of DOTAP, DODMA, DOPC, CHOL, and PEG-DPPE present inrespective molar ratios of 15:25:36:20:4; and, inhibiting miRNA activityin vitro or in vivo.
 2. The method of claim 1, wherein theoligonucleotide comprises one of compounds 101-119, having SEQ ID NOs.4-22, respectively.
 3. The method of claim 1, wherein theoligonucleotide is a synthetic oligonucleotide.
 4. The method of claim1, wherein the miRNA is miR-21.
 5. The method of claim 1, wherein theoligonucleotide is an anti-miR-21 oligonucleotide.
 6. The method ofclaim 1, wherein the oligonucleotide comprises an anti-miR-21oligonucleotide selected from the group consisting of SEQ ID NO: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 23, 24, 25, 26, 27 and
 28. 7. The method of claim 1, wherein theoligonucleotide is an anti-miR-21 oligonucleotide that comprisescompound 116 having SEQ ID NO:19, or compound 117 having SEQ ID NO:20.8. The method of claim 1, wherein the oligonucleotide is an anti-miR-21oligonucleotide having SEQ ID NO:3.
 9. The method of claim 1, whereinthe lipid nanoparticles are coformulated with one or more peptidesselected from the group consisting of gramicidin A, B, C, D, and S; HA2;JTS-1; proteinase K (PrK); trichorovin-Xlla (TV-Xlla); rabies virusglycoprotein (RVG); interleukin-1 ˜; HIV-Tat; herpes simplex virus(HSV), and VP22 protein.
 10. The method of claim 1, wherein inhibitingmiRNA activity comprises lowering the activity of the miRNA in vivo in asubject, by contacting a biological sample expressing miR-21 thereinwith the inhibitor composition.
 11. The method of claim 10 wherein thebiological sample is a cancer cell which over-expresses miR-21 relativeto non-tumor cells.
 12. The method of claim 1, wherein the miRNAactivity is measured as the expression levels of miR-21 target genesselected from: ANKRD46, PTEN, PDCD4, DDAH1, RECK and/or TIMP3.
 13. Themethod of claim 1, further including administering at least oneadditional therapeutic agent to the subject prior to, simultaneous with,or subsequent to, administration of the inhibitor composition.
 14. Themethod of claim 13, wherein the therapeutic agent comprises paclitaxel.15. The method of claim 1, wherein the subject has one or more of lungcancer, ovarian cancer, breast cancer, or a glioma.
 16. The method ofclaim 1, wherein the tertiary amine-cationic lipids further include oneor more of: DODAP, DC-CHOL, N,N-dimethylhexadecylamine, or combinationsthereof; and, the quaternary amine-cationic lipids further include oneor more of: DOTMA, DDAB, or combinations thereof.
 17. A method oftreating a condition characterized by over-expression of miRNAcomprising, administering an inhibitor composition to a subject at aconcentration sufficient to inhibit the action of the miRNA; wherein thecomposition comprises at least one oligonucleotide capable of inhibitingactivity of the miRNA encapsulated in the lipid nanoparticle of claim 1.